Insecticidal proteins and methods for their use

ABSTRACT

Compositions and methods for controlling pests are provided. The methods involve transforming organisms with a nucleic acid sequence encoding an insecticidal protein. In particular, the nucleic acid sequences are useful for preparing plants and microorganisms that possess insecticidal activity. Thus, transformed bacteria, plants, plant cells, plant tissues and seeds are provided. Compositions are insecticidal nucleic acids and proteins of bacterial species. The sequences find use in the construction of expression vectors for subsequent transformation into organisms of interest including plants, as probes for the isolation of other homologous (or partially homologous) genes. The pesticidal proteins find use in controlling, inhibiting growth or killing Lepidopteran, Coleopteran, Dipteran, fungal, Hemipteran and nematode pest populations and for producing compositions with insecticidal activity.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/438,179 filed on Dec. 22, 2016, which is incorporated herein byreference in its entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The official copy of the sequence listing is submitted electronicallyvia EFS-Web as an ASCII formatted sequence listing with a file named“6729WOPCT_Sequence_Listing” created on Nov. 30, 2017, and having a sizeof 107 kilobytes and is filed concurrently with the specification. Thesequence listing contained in this ASCII formatted document is part ofthe specification and is herein incorporated by reference in itsentirety.

FIELD

This disclosure relates to the field of molecular biology. Provided arenovel genes that encode pesticidal proteins. These pesticidal proteinsand the nucleic acid sequences that encode them are useful in preparingpesticidal formulations and in the production of transgenicpest-resistant plants.

BACKGROUND

Biological control of insect pests of agricultural significance using amicrobial agent, such as fungi, bacteria or another species of insectaffords an environmentally friendly and commercially attractivealternative to synthetic chemical pesticides. Generally speaking, theuse of biopesticides presents a lower risk of pollution andenvironmental hazards and biopesticides provide greater targetspecificity than is characteristic of traditional broad-spectrumchemical insecticides. In addition, biopesticides often cost less toproduce and thus improve economic yield for a wide variety of crops.

Certain species of microorganisms of the genus Bacillus are known topossess pesticidal activity against a range of insect pests includingLepidoptera, Diptera, Coleoptera, Hemiptera and others. Bacillusthuringiensis (Bt) and Bacillus popilliae are among the most successfulbiocontrol agents discovered to date. Insect pathogenicity has also beenattributed to strains of B. larvae, B. lentimorbus, B. sphaericus and B.cereus. Microbial insecticides, particularly those obtained fromBacillus strains, have played an important role in agriculture asalternatives to chemical pest control.

Crop plants have been developed with enhanced insect resistance bygenetically engineering crop plants to produce pesticidal proteins fromBacillus. For example, corn and cotton plants have been geneticallyengineered to produce pesticidal proteins isolated from strains ofBacillus thuringiensis. These genetically engineered crops are nowwidely used in agriculture and have provided the farmer with anenvironmentally friendly alternative to traditional insect-controlmethods. While they have proven to be very successful commercially,these genetically engineered, insect-resistant crop plants may provideresistance to only a narrow range of the economically important insectpests. In some cases, insects can develop resistance to differentinsecticidal compounds, which raises the need to identify alternativebiological control agents for pest control.

Accordingly, there remains a need for new pesticidal proteins withdifferent ranges of insecticidal activity against insect pests, e.g.,insecticidal proteins which are active against a variety of insects inthe order Lepidoptera and the order Coleoptera, including but notlimited to insect pests that have developed resistance to existinginsecticides.

SUMMARY

In one aspect compositions and methods for conferring pesticidalactivity to bacteria, plants, plant cells, tissues and seeds areprovided. Compositions include nucleic acid molecules encoding sequencesfor pesticidal and insecticidal polypeptides, vectors comprising thosenucleic acid molecules, and host cells comprising the vectors.Compositions also include the pesticidal polypeptide sequences andantibodies to those polypeptides. Compositions also comprise transformedbacteria, plants, plant cells, tissues and seeds.

In another aspect isolated or recombinant nucleic acid molecules areprovided encoding IPD101 polypeptides including amino acidsubstitutions, deletions, insertions, and fragments thereof. Providedare isolated or recombinant nucleic acid molecules capable of encodingIPD101 polypeptides of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20,22, 24, 25, 26, 28, 29, 30, 32, 46, 48, 50, 52, 54, 56, 58, and 60, aswell as amino acid substitutions, deletions, insertions, fragmentsthereof, and combinations thereof. Nucleic acid sequences that arecomplementary to a nucleic acid sequence of the embodiments or thathybridize to a sequence of the embodiments are also encompassed. Thenucleic acid sequences can be used in DNA constructs or expressioncassettes for transformation and expression in organisms, includingmicroorganisms and plants. The nucleotide or amino acid sequences may besynthetic sequences that have been designed for expression in anorganism including, but not limited to, a microorganism or a plant.

In another aspect IPD101 polypeptides are encompassed. Also provided areisolated or recombinant IPD101 polypeptides of SEQ ID NO: 2, 4, 6, 8,10, 12, 14, 16, 18, 20, 22, 24, 25, 26, 28, 29, 30, 32, 46, 48, 50, 52,54, 56, 58, and 60, as well as amino acid substitutions, deletions,insertions, fragments thereof and combinations thereof.

In another aspect methods are provided for producing the polypeptidesand for using those polypeptides for controlling or killing aLepidopteran, Coleopteran, nematode, fungi, and/or Dipteran pests. Thetransgenic plants of the embodiments express one or more of thepesticidal sequences disclosed herein. In various embodiments, thetransgenic plant further comprises one or more additional genes forinsect resistance, for example, one or more additional genes forcontrolling Coleopteran, Lepidopteran, Hemipteran or nematode pests. Itwill be understood by one of skill in the art that the transgenic plantmay comprise any gene imparting an agronomic trait of interest.

In another aspect methods for detecting the nucleic acids andpolypeptides of the embodiments in a sample are also included. A kit fordetecting the presence of an IPD101 polypeptide or detecting thepresence of a polynucleotide encoding an IPD101 polypeptide in a sampleis provided. The kit may be provided along with all reagents and controlsamples necessary for carrying out a method for detecting the intendedagent, as well as instructions for use.

In another aspect the compositions and methods of the embodiments areuseful for the production of organisms with enhanced pest resistance ortolerance. These organisms and compositions comprising the organisms aredesirable for agricultural purposes. The compositions of the embodimentsare also useful for generating altered or improved proteins that havepesticidal activity or for detecting the presence of IPD101polypeptides.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1(a)-(d) shows an amino acid sequence alignment, using the ALIGNX®module of the Vector NTI® suite, of the IPD101Aa polypeptide (SEQ ID NO:2), the IPD101Ab polypeptide (SEQ ID NO: 4), the IPD101Ac polypeptide(SEQ ID NO: 6), the IPD101Ba polypeptide (SEQ ID NO: 8), the IPD101Capolypeptide (SEQ ID NO: 10), the IPD101Cb polypeptide (SEQ ID NO: 12),the IPD101Cc polypeptide (SEQ ID NO: 14), the IPD101Cd polypeptide (SEQID NO: 16), the IPD101Ce polypeptide (SEQ ID NO: 18), the IPD101Cfpolypeptide (SEQ ID NO: 20), the IPD101Ea polypeptide (SEQ ID NO: 22),the IPD101Eb polypeptide (SEQ ID NO: 24), the IPD101Ee polypeptide (SEQID NO: 25), the IPD101Fa polypeptide (SEQ ID NO: 26), the IPD101Fbpolypeptide (SEQ ID NO: 28), the IPD101Ga polypeptide (SEQ ID NO: 29),the IPD101Gb polypeptide (SEQ ID NO: 30), the IPD101Gc polypeptide (SEQID NO: 32), the IPD101Gd polypeptide (SEQ ID NO: 56), the IPD101Gepolypeptide (SEQ ID NO: 58), and the IPD101Gf polypeptide (SEQ ID NO:60). The amino acid sequence diversity between the amino acid sequencesis highlighted. Conservative amino acid differences are indicated by (

) shading.

FIG. 2: Homologous competition of Alexa-labeled IPD101Aa (1.5 nM)binding to WCRW BBMVs reveals specific binding with high apparentaffinity (EC50=2 nM).

DETAILED DESCRIPTION

It is to be understood that this disclosure is not limited to theparticular methodology, protocols, cell lines, genera, and reagentsdescribed, as such may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentdisclosure.

As used herein the singular forms “a”, “and”, and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to “a cell” includes a plurality of such cells andreference to “the protein” includes reference to one or more proteinsand equivalents thereof, and so forth. All technical and scientificterms used herein have the same meaning as commonly understood to one ofordinary skill in the art to which this disclosure belongs unlessclearly indicated otherwise.

The present disclosure is drawn to compositions and methods forcontrolling pests. The methods involve transforming organisms withnucleic acid sequences encoding IPD101 polypeptides. In particular, thenucleic acid sequences of the embodiments are useful for preparingplants and microorganisms that possess pesticidal activity. Thus,transformed bacteria, plants, plant cells, plant tissues and seeds areprovided. The compositions include pesticidal nucleic acids and proteinsof bacterial species. The nucleic acid sequences find use in theconstruction of expression vectors for subsequent transformation intoorganisms of interest, as probes for the isolation of other homologous(or partially homologous) genes, and for the generation of alteredIPD101 polypeptides by methods known in the art, such as site directedmutagenesis, domain swapping or DNA shuffling. The IPD101 polypeptidesfind use in controlling or killing Lepidopteran, Coleopteran, Dipteran,fungal, Hemipteran and nematode pest populations and for producingcompositions with pesticidal activity. Insect pests of interest include,but are not limited to, Lepidoptera species including but not limitedto: Corn Earworm, (CEW) (Helicoverpa zea), European Corn Borer (ECB)(Ostrinia nubialis), diamond-back moth, e.g., Helicoverpa zea Boddie;soybean looper, e.g., Pseudoplusia includens Walker; and velvet beancaterpillar e.g., Anticarsia gemmatalis Hubner and Coleoptera speciesincluding but not limited to Western corn rootworm (Diabroticavirgifera)—WCRW, Southern corn rootworm (Diabrotica undecimpunctatahowardi)—SCRW, and Northern corn rootworm (Diabrotica barberi)—NCRW.

By “pesticidal toxin” or “pesticidal protein” is used herein to refer toa toxin that has toxic activity against one or more pests, including,but not limited to, members of the Lepidoptera, Diptera, Hemiptera andColeoptera orders or the Nematoda phylum or a protein that has homologyto such a protein. Pesticidal proteins have been isolated from organismsincluding, for example, Bacillus sp., Pseudomonas sp., Photorhabdus sp.,Xenorhabdus sp., Clostridium bifermentans and Paenibacillus popilliae.

In some embodiments the IPD101 polypeptide includes an amino acidsequence deduced from the full-length nucleic acid sequence disclosedherein and amino acid sequences that are shorter than the full-lengthsequences, either due to the use of an alternate downstream start siteor due to processing that produces a shorter protein having pesticidalactivity. Processing may occur in the organism the protein is expressedin or in the pest after ingestion of the protein.

Thus, provided herein are novel isolated or recombinant nucleic acidsequences that confer pesticidal activity. Also provided are the aminoacid sequences of IPD101 polypeptides. The polypeptides resulting fromtranslation of these IPD101 genes allows cells to control or kill peststhat ingest it.

IPD101 Proteins and Variants and Fragments Thereof

IPD101 polypeptides are encompassed by the disclosure. “IPD101polypeptide”, and “IPD101 protein” as used herein interchangeably refersto a polypeptide(s) having insecticidal activity including but notlimited to insecticidal activity against one or more insect pests of theLepidoptera and/or Coleoptera orders, and is sufficiently homologous tothe IPD101Aa polypeptide of SEQ ID NO: 2. A variety of IPD101polypeptides are contemplated. Sources of IPD101 polypeptides or relatedproteins include bacterial species selected from but not limited toLysinibacillus species. Alignment of the amino acid sequences of IPD101polypeptide homologs (for example, see FIG. 1), allows for theidentification of residues that are highly conserved amongst the naturalhomologs of this family.

“Sufficiently homologous” is used herein to refer to an amino acidsequence that has at least about 40%, 45%, 50%, 51%, 52%, 53%, 54%, 55%,56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%,70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or greater sequence homology compared to a reference sequenceusing one of the alignment programs described herein using standardparameters. In some embodiments the sequence homology is against thefull length sequence of an IPD101 polypeptide. In some embodiments theIPD101 polypeptide has at least about 40%, 45%, 50%, 51%, 52%, 53%, 54%,55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%,69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or greater sequence identity compared to any one of SEQ IDNOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 25, 26, 28, 29, 30, 32,46, 48, 50, 52, 54, 56, 58, and 60. The term “about” when used herein incontext with percent sequence identity means+/−0.5%. One of skill in theart will recognize that these values can be appropriately adjusted todetermine corresponding homology of proteins taking into account aminoacid similarity and the like. In some embodiments the sequence identityis calculated using ClustalW algorithm in the ALIGNX® module of theVector NTI® Program Suite (Invitrogen Corporation, Carlsbad, Calif.)with all default parameters. In some embodiments the sequence identityis across the entire length of polypeptide calculated using ClustalWalgorithm in the ALIGNX® module of the Vector NTI® Program Suite(Invitrogen Corporation, Carlsbad, Calif.) with all default parameters.

As used herein, the terms “protein,” “peptide molecule,” or“polypeptide” includes any molecule that comprises five or more aminoacids. It is well known in the art that protein, peptide or polypeptidemolecules may undergo modification, including post-translationalmodifications, such as, but not limited to, disulfide bond formation,glycosylation, phosphorylation or oligomerization. Thus, as used herein,the terms “protein,” “peptide molecule” or “polypeptide” includes anyprotein that is modified by any biological or non-biological process.The terms “amino acid” and “amino acids” refer to all naturallyoccurring L-amino acids.

A “recombinant protein” is used herein to refer to a protein that is nolonger in its natural environment, for example in vitro or in arecombinant bacterial or plant host cell. An IPD101 polypeptide that issubstantially free of cellular material includes preparations of proteinhaving less than about 30%, 20%, 10% or 5% (by dry weight) ofnon-pesticidal protein (also referred to herein as a “contaminatingprotein”).

“Fragments” or “biologically active portions” include polypeptidefragments comprising amino acid sequences sufficiently identical to anIPD101 polypeptide and that exhibit insecticidal activity. “Fragments”or “biologically active portions” of IPD101 polypeptides includesfragments comprising amino acid sequences sufficiently identical to theamino acid sequence set forth in any one of SEQ ID NOS: 2, 4, 6, 8, 10,12, 14, 16, 18, 20, 22, 24, 25, 26, 28, 29, 30, 32, 46, 48, 50, 52, 54,56, 58, and 60 wherein the IPD101 polypeptide has insecticidal activity.Such biologically active portions can be prepared by recombinanttechniques and evaluated for insecticidal activity. In some embodiments,the IPD101 polypeptide fragment is an N-terminal and/or a C-terminaltruncation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 ormore amino acids from the N-terminus and/or C-terminus relative to anyone of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 25, 26,28, 29, 30, 32, 46, 48, 50, 52, 54, 56, 58, and 60, e.g., byproteolysis, by insertion of a start codon, by deletion of the codonsencoding the deleted amino acids and concomitant insertion of a startcodon, and/or insertion of a stop codon. In some embodiments, the IPD101polypeptide fragment is an N-terminal truncation of at least 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24 amino acids from the N-terminus of any one of SEQ ID NOS: 2, 4, 6, 8,10, 12, 14, 16, 18, 20, 22, 24, 25, 26, 28, 29, 30, 32, 46, 48, 50, 52,54, 56, 58, and 60. In some embodiments, the IPD101 polypeptide fragmentis an N-terminal and/or a C-terminal truncation of at least 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or more amino acids from theN-terminus and/or C-terminus relative to any one of SEQ ID NOS: 2, 4, 6,8, 10, 12, 14, 16, 18, 20, 22, 24, 25, 26, 28, 29, 30, 32, 46, 48, 50,52, 54, 56, 58, and 60.

“Variants” as used herein refers to proteins or polypeptides having anamino acid sequence that is at least about 50%, 55%, 60%, 65%, 70%, 75%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or greater identical to the parental aminoacid sequence.

In some embodiments an IPD101 polypeptide comprises an amino acidsequence having at least about 40%, 45%, 50%, 51%, 52%, 53%, 54%, 55%,56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%,70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or greater identity to the amino acid sequence of any one ofSEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 25, 26, 28, 29,30, 32, 46, 48, 50, 52, 54, 56, 58, and 60, wherein the IPD101polypeptide has insecticidal activity.

In some embodiments an IPD101 polypeptide comprises an amino acidsequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greateridentity across the entire length of the amino acid sequence of any oneof SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 25, 26, 28,29, 30, 32, 46, 48, 50, 52, 54, 56, 58, and 60.

In some embodiments the sequence identity is across the entire length ofthe polypeptide calculated using ClustalW algorithm in the ALIGNX®module of the Vector NTI® Program Suite (Invitrogen Corporation,Carlsbad, Calif.) with all default parameters.

In some embodiments an IPD101 polypeptide comprises an amino acidsequence of any one or more of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16,18, 20, 22, 24, 25, 26, 28, 29, 30, 32, 46, 48, 50, 52, 54, 56, 58, and60 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,91, 92, 93, 94, 95 or more amino acid substitutions compared to thenative amino acid at the corresponding position of any one or more ofthe respective SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,25, 26, 28, 29, 30, 32, 46, 48, 50, 52, 54, 56, 58, and 60.

Methods for such manipulations are generally known in the art. Forexample, amino acid sequence variants of an IPD101 polypeptide can beprepared by mutations in the DNA. This may also be accomplished by oneof several forms of mutagenesis and/or in directed evolution. In someaspects, the changes encoded in the amino acid sequence will notsubstantially affect the function of the protein. Such variants willpossess the desired pesticidal activity. However, it is understood thatthe ability of an IPD101 polypeptide to confer pesticidal activity maybe improved by the use of such techniques upon the compositions of thisdisclosure.

For example, conservative amino acid substitutions may be made at one ormore predicted nonessential amino acid residues. A “nonessential” aminoacid residue is a residue that can be altered from the wild-typesequence of an IPD101 polypeptide without altering the biologicalactivity. A “conservative amino acid substitution” is one in which theamino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families include: amino acidswith basic side chains (e.g., lysine, arginine, histidine); acidic sidechains (e.g., aspartic acid, glutamic acid); polar, negatively chargedresidues and their amides (e.g., aspartic acid, asparagine, glutamic,acid, glutamine; uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine); small aliphatic,nonpolar or slightly polar residues (e.g., Alanine, serine, threonine,proline, glycine); nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan); largealiphatic, nonpolar residues (e.g., methionine, leucine, isoleucine,valine, cystine); beta-branched side chains (e.g., threonine, valine,isoleucine); aromatic side chains (e.g., tyrosine, phenylalanine,tryptophan, histidine); large aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan).

Amino acid substitutions may be made in nonconserved regions that retainfunction. In general, such substitutions would not be made for conservedamino acid residues or for amino acid residues residing within aconserved motif, where such residues are essential for protein activity.Examples of residues that are conserved and that may be essential forprotein activity include, for example, residues that are identicalbetween all proteins contained in an alignment of similar or relatedtoxins to the sequences of the embodiments (e.g., residues that areidentical in an alignment of homologous proteins). Examples of residuesthat are conserved but that may allow conservative amino acidsubstitutions and still retain activity include, for example, residuesthat have only conservative substitutions between all proteins containedin an alignment of similar or related toxins to the sequences of theembodiments (e.g., residues that have only conservative substitutionsbetween all proteins contained in the alignment homologous proteins).However, one of skill in the art would understand that functionalvariants may have minor conserved or nonconserved alterations in theconserved residues. Guidance as to appropriate amino acid substitutionsthat do not affect biological activity of the protein of interest may befound in the model of Dayhoff, et al., (1978) Atlas of Protein Sequenceand Structure (Natl. Biomed. Res. Found., Washington, D.C.).

In making such changes, the hydropathic index of amino acids may beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte and Doolittle, (1982) J Mol Biol.157(1):105-32). It is accepted that the relative hydropathic characterof the amino acid contributes to the secondary structure of theresultant protein, which in turn defines the interaction of the proteinwith other molecules, for example, enzymes, substrates, receptors, DNA,antibodies, antigens, and the like.

It is known in the art that certain amino acids may be substituted byother amino acids having a similar hydropathic index or score and stillresult in a protein with similar biological activity, i.e., still obtaina biological functionally equivalent protein. Each amino acid has beenassigned a hydropathic index on the basis of its hydrophobicity andcharge characteristics (Kyte and Doolittle, ibid). These are: isoleucine(+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8);cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine(−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine(−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine(−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9) and arginine(−4.5). In making such changes, the substitution of amino acids whosehydropathic indices are within +2 is preferred, those which are within+1 are particularly preferred, and those within +0.5 are even moreparticularly preferred.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity. U.S. Pat.No. 4,554,101, states that the greatest local average hydrophilicity ofa protein, as governed by the hydrophilicity of its adjacent aminoacids, correlates with a biological property of the protein.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0.+0.1); glutamate (+3.0.+0.1); serine(+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine(−0.4); proline (−0.5.+0.1); alanine (−0.5); histidine (−0.5); cysteine(−1.0); methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine(−1.8); tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4).

Alternatively, alterations may be made to the protein sequence of manyproteins at the amino or carboxy terminus without substantiallyaffecting activity. This can include insertions, deletions oralterations introduced by modern molecular methods, such as PCR,including PCR amplifications that alter or extend the protein codingsequence by virtue of inclusion of amino acid encoding sequences in theoligonucleotides utilized in the PCR amplification. Alternatively, theprotein sequences added can include entire protein-coding sequences,such as those used commonly in the art to generate protein fusions. Suchfusion proteins are often used to (1) increase expression of a proteinof interest (2) introduce a binding domain, enzymatic activity orepitope to facilitate either protein purification, protein detection orother experimental uses known in the art (3) target secretion ortranslation of a protein to a subcellular organelle, such as theperiplasmic space of Gram-negative bacteria, mitochondria orchloroplasts of plants or the endoplasmic reticulum of eukaryotic cells,the latter of which often results in glycosylation of the protein.

Variant nucleotide and amino acid sequences of the disclosure alsoencompass sequences derived from mutagenic and recombinogenic proceduressuch as DNA shuffling. With such a procedure, one or more differentIPD101 polypeptide coding regions can be used to create a new IPD101polypeptide possessing the desired properties. In this manner, librariesof recombinant polynucleotides are generated from a population ofrelated sequence polynucleotides comprising sequence regions that havesubstantial sequence identity and can be homologously recombined invitro or in vivo. For example, using this approach, sequence motifsencoding a domain of interest may be shuffled between a pesticidal geneand other known pesticidal genes to obtain a new gene coding for aprotein with an improved property of interest, such as an increasedinsecticidal activity. Strategies for such DNA shuffling are known inthe art. See, for example, Stemmer, (1994) Proc. Natl. Acad. Sci. USA91:10747-10751; Stemmer, (1994) Nature 370:389-391; Crameri, et al.,(1997) Nature Biotech. 15:436-438; Moore, et al., (1997) J. Mol. Biol.272:336-347; Zhang, et al., (1997) Proc. Natl. Acad. Sci. USA94:4504-4509; Crameri, et al., (1998) Nature 391:288-291; and U.S. Pat.Nos. 5,605,793 and 5,837,458.

Domain swapping or shuffling is another mechanism for generating alteredIPD101 polypeptides. Domains may be swapped between IPD101 polypeptidesresulting in hybrid or chimeric toxins with improved insecticidalactivity or target spectrum. Methods for generating recombinant proteinsand testing them for pesticidal activity are well known in the art (see,for example, Naimov, et al., (2001) Appl. Environ. Microbiol.67:5328-5330; de Maagd, et al., (1996) Appl. Environ. Microbiol.62:1537-1543; Ge, et al., (1991) J. Biol. Chem. 266:17954-17958;Schnepf, et al., (1990) J. Biol. Chem. 265:20923-21010; Rang, et al.,91999) Appl. Environ. Microbiol. 65:2918-2925).

Phylogenetic, Sequence Motif, and Structural Analyses of InsecticidalProtein Families.

A sequence and structure analysis method can be employed, which iscomposed of four components: phylogenetic tree construction, proteinsequence motifs finding, secondary structure prediction, and alignmentof protein sequences and secondary structures. Details about eachcomponent are illustrated below.

1) Phylogenetic Tree Construction

The phylogenetic analysis can be performed using the software MEGA5.Protein sequences can be subjected to ClustalW version 2 analysis(Larkin M. A et al (2007) Bioinformatics 23(21): 2947-2948) for multiplesequence alignment. The evolutionary history is then inferred by theMaximum Likelihood method based on the JTT matrix-based model. The treewith the highest log likelihood is obtained, exported in Newick format,and further processed to extract the sequence IDs in the same order asthey appeared in the tree. A few clades representing sub-families can bemanually identified for each insecticidal protein family.

2) Protein Sequence Motifs Finding

Protein sequences are re-ordered according to the phylogenetic treebuilt previously, and fed to the MOTIF analysis tool MEME (Multiple EMfor MOTIF Elicitation) (Bailey T. L., and Elkan C., Proceedings of theSecond International Conference on Intelligent Systems for MolecularBiology, pp. 28-36, AAAI Press, Menlo Park, Calif., 1994.) foridentification of key sequence motifs. MEME is setup as follows: Minimumnumber of sites 2, Minimum motif width 5, and Maximum number of motifs30. Sequence motifs unique to each sub-family were identified by visualobservation. The distribution of MOTIFs across the entire gene familycould be visualized in HTML webpage. The MOTIFs are numbered relative tothe ranking of the E-value for each MOTIF.

3) Secondary Structure Prediction

PSIPRED, top ranked secondary structure prediction method (Jones D T.(1999) J. Mol. Biol. 292: 195-202), can be used for protein secondarystructure prediction. The tool provides accurate structure predictionusing two feed-forward neural networks based on the PSI-BLAST output.The PSI-BLAST database is created by removing low-complexity,transmembrane, and coiled-coil regions in Uniref100. The PSIPRED resultscontain the predicted secondary structures (Alpha helix: H, Beta strand:E, and Coil: C) and the corresponding confidence scores for each aminoacid in a given protein sequence.

4) Alignment of Protein Sequences and Secondary Structures

A script can be developed to generate gapped secondary structurealignment according to the multiple protein sequence alignment from step1 for all proteins. All aligned protein sequences and structures areconcatenated into a single FASTA file, and then imported into MEGA forvisualization and identification of conserved structures.

In some embodiments the IPD101 polypeptide has a modified physicalproperty. As used herein, the term “physical property” refers to anyparameter suitable for describing the physical-chemical characteristicsof a protein. As used herein, “physical property of interest” and“property of interest” are used interchangeably to refer to physicalproperties of proteins that are being investigated and/or modified.Examples of physical properties include, but are not limited to, netsurface charge and charge distribution on the protein surface, nethydrophobicity and hydrophobic residue distribution on the proteinsurface, surface charge density, surface hydrophobicity density, totalcount of surface ionizable groups, surface tension, protein size and itsdistribution in solution, melting temperature, heat capacity, and secondvirial coefficient. Examples of physical properties also include, IPD101polypeptide having increased expression, increased solubility, decreasedphytotoxicity, and digestibility of proteolytic fragments in an insectgut. Models for digestion by simulated gastric fluids are known to oneskilled in the art (Fuchs, R. L. and J. D. Astwood. Food Technology 50:83-88, 1996; Astwood, J. D., et al Nature Biotechnology 14: 1269-1273,1996; Fu T J et al J. Agric Food Chem. 50: 7154-7160, 2002).

In some embodiments variants include polypeptides that differ in aminoacid sequence due to mutagenesis. Variant proteins encompassed by thedisclosure are biologically active, that is they continue to possess thedesired biological activity (i.e. pesticidal activity) of the nativeprotein. In some embodiment the variant will have at least about 10%, atleast about 30%, at least about 50%, at least about 70%, at least about80% or more of the insecticidal activity of the native protein. In someembodiments, the variants may have improved activity over the nativeprotein.

Bacterial genes quite often possess multiple methionine initiationcodons in proximity to the start of the open reading frame. Often,translation initiation at one or more of these start codons will lead togeneration of a functional protein. These start codons can include ATGcodons. However, bacteria such as Bacillus sp. also recognize the codonGTG as a start codon, and proteins that initiate translation at GTGcodons contain a methionine at the first amino acid. On rare occasions,translation in bacterial systems can initiate at a TTG codon, though inthis event the TTG encodes a methionine. Furthermore, it is not oftendetermined a priori which of these codons are used naturally in thebacterium. Thus, it is understood that use of one of the alternatemethionine codons may also lead to generation of pesticidal proteins.These pesticidal proteins are encompassed in the present disclosure andmay be used in the methods of the present disclosure. It will beunderstood that, when expressed in plants, it will be necessary to alterthe alternate start codon to ATG for proper translation.

In some embodiments an IPD101 polypeptide comprises the amino acidsequence of any one or more of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16,18, 20, 22, 24, 25, 26, 28, 29, 30, 32, 46, 48, 50, 52, 54, 56, 58, and60.

In some embodiments, chimeric polypeptides are provided comprisingregions of at least two different IPD101 polypeptides of the disclosure.

In some embodiments, chimeric polypeptides are provided comprisingregions of at least two different IPD101 polypeptides selected from anyone or more of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,25, 26, 28, 29, 30, 32, 46, 48, 50, 52, 54, 56, 58, and 60.

In some embodiments, chimeric IPD101 polypeptide(s) are providedcomprising an N-terminal Region of a first IPD101 polypeptide of thedisclosure operably fused to a C-terminal Region of a second IPD101polypeptide of the disclosure.

In other embodiments the IPD101 polypeptide may be expressed as aprecursor protein with an intervening sequence that catalyzesmulti-step, post translational protein splicing. Protein splicinginvolves the excision of an intervening sequence from a polypeptide withthe concomitant joining of the flanking sequences to yield a newpolypeptide (Chong, et al., (1996) J. Biol. Chem., 271:22159-22168).This intervening sequence or protein splicing element, referred to asinteins, which catalyze their own excision through three coordinatedreactions at the N-terminal and C-terminal splice junctions: an acylrearrangement of the N-terminal cysteine or serine; a transesterficationreaction between the two termini to form a branched ester or thioesterintermediate and peptide bond cleavage coupled to cyclization of theintein C-terminal asparagine to free the intein (Evans, et al., (2000)J. Biol. Chem., 275:9091-9094). The elucidation of the mechanism ofprotein splicing has led to a number of intein-based applications (Comb,et al., U.S. Pat. No. 5,496,714; Comb, et al., U.S. Pat. No. 5,834,247;Camarero and Muir, (1999) J. Amer. Chem. Soc. 121:5597-5598; Chong, etal., (1997) Gene 192:271-281, Chong, et al., (1998) Nucleic Acids Res.26:5109-5115; Chong, et al., (1998) J. Biol. Chem. 273:10567-10577;Cotton, et al., (1999) J. Am. Chem. Soc. 121:1100-1101; Evans, et al.,(1999) J. Biol. Chem. 274:18359-18363; Evans, et al., (1999) J. Biol.Chem. 274:3923-3926; Evans, et al., (1998) Protein Sci. 7:2256-2264;Evans, et al., (2000) J. Biol. Chem. 275:9091-9094; Iwai and Pluckthun,(1999) FEBS Lett. 459:166-172; Mathys, et al., (1999) Gene 231:1-13;Mills, et al., (1998) Proc. Natl. Acad. Sci. USA 95:3543-3548; Muir, etal., (1998) Proc. Natl. Acad. Sci. USA 95:6705-6710; Otomo, et al.,(1999) Biochemistry 38:16040-16044; Otomo, et al., (1999) J. Biolmol.NMR 14:105-114; Scott, et al., (1999) Proc. Natl. Acad. Sci. USA96:13638-13643; Severinov and Muir, (1998) J. Biol. Chem.273:16205-16209; Shingledecker, et al., (1998) Gene 207:187-195;Southworth, et al., (1998) EMBO J. 17:918-926; Southworth, et al.,(1999) Biotechniques 27:110-120; Wood, et al., (1999) Nat. Biotechnol.17:889-892; Wu, et al., (1998a) Proc. Natl. Acad. Sci. USA 95:9226-9231;Wu, et al., (1998b) Biochim Biophys Acta 1387:422-432; Xu, et al.,(1999) Proc. Natl. Acad. Sci. USA 96:388-393; Yamazaki, et al., (1998)J. Am. Chem. Soc., 120:5591-5592). For the application of inteins inplant transgenes, see, Yang, et al., (Transgene Res 15:583-593 (2006))and Evans, et al., (Annu. Rev. Plant Biol. 56:375-392 (2005)).

In another embodiment the IPD101 polypeptide may be encoded by twoseparate genes where the intein of the precursor protein comes from thetwo genes, referred to as a split-intein, and the two portions of theprecursor are joined by a peptide bond formation. This peptide bondformation is accomplished by intein-mediated trans-splicing. For thispurpose, a first and a second expression cassette comprising the twoseparate genes further code for inteins capable of mediating proteintrans-splicing. By trans-splicing, the proteins and polypeptides encodedby the first and second fragments may be linked by peptide bondformation. Trans-splicing inteins may be selected from the nucleolar andorganellar genomes of different organisms including eukaryotes,archaebacteria and eubacteria. Inteins that may be used for are listedat neb.com/neb/inteins.html, which can be accessed on the world-wide webusing the “www” prefix). The nucleotide sequence coding for an inteinmay be split into a 5′ and a 3′ part that code for the 5′ and the 3′part of the intein, respectively. Sequence portions not necessary forintein splicing (e.g. homing endonuclease domain) may be deleted. Theintein coding sequence is split such that the 5′ and the 3′ parts arecapable of trans-splicing. For selecting a suitable splitting site ofthe intein coding sequence, the considerations published by Southworth,et al., (1998) EMBO J. 17:918-926 may be followed. In constructing thefirst and the second expression cassette, the 5′ intein coding sequenceis linked to the 3′ end of the first fragment coding for the N-terminalpart of the IPD101 polypeptide and the 3′ intein coding sequence islinked to the 5′ end of the second fragment coding for the C-terminalpart of the IPD101 polypeptide.

In general, the trans-splicing partners can be designed using any splitintein, including any naturally-occurring or artificially-split splitintein. Several naturally-occurring split inteins are known, forexample: the split intein of the DnaE gene of Synechocystis sp. PCC6803(see, Wu, et al., (1998) Proc Natl Acad Sci USA. 95(16):9226-31 andEvans, et al., (2000) J Biol Chem. 275(13):9091-4 and of the DnaE genefrom Nostoc punctiforme (see, Iwai, et al., (2006) FEBS Lett.580(7):1853-8). Non-split inteins have been artificially split in thelaboratory to create new split inteins, for example: the artificiallysplit Ssp DnaB intein (see, Wu, et al., (1998) Biochim Biophys Acta.1387:422-32) and split Sce VMA intein (see, Brenzel, et al., (2006)Biochemistry. 45(6):1571-8) and an artificially split fungal mini-intein(see, Elleuche, et al., (2007) Biochem Biophys Res Commun.355(3):830-4). There are also intein databases available that catalogueknown inteins (see for example the online-database available at:bioinformatics.weizmann.ac.il/^(˜)pietro/inteins/Inteinstable.html,which can be accessed on the world-wide web using the “www” prefix).

Naturally-occurring non-split inteins may have endonuclease or otherenzymatic activities that can typically be removed when designing anartificially-split split intein. Such mini-inteins or minimized splitinteins are well known in the art and are typically less than 200 aminoacid residues long (see, Wu, et al., (1998) Biochim Biophys Acta.1387:422-32). Suitable split inteins may have other purificationenabling polypeptide elements added to their structure, provided thatsuch elements do not inhibit the splicing of the split intein or areadded in a manner that allows them to be removed prior to splicing.Protein splicing has been reported using proteins that comprisebacterial intein-like (BIL) domains (see, Amitai, et al., (2003) MolMicrobiol. 47:61-73) and hedgehog (Hog) auto-processing domains (thelatter is combined with inteins when referred to as the Hog/inteinsuperfamily or HINT family (see, Dassa, et al., (2004) J Biol Chem.279:32001-7) and domains such as these may also be used to prepareartificially-split inteins. In particular, non-splicing members of suchfamilies may be modified by molecular biology methodologies to introduceor restore splicing activity in such related species. Recent studiesdemonstrate that splicing can be observed when a N-terminal split inteincomponent is allowed to react with a C-terminal split intein componentnot found in nature to be its “partner”; for example, splicing has beenobserved utilizing partners that have as little as 30 to 50% homologywith the “natural” splicing partner (see, Dassa, et al., (2007)Biochemistry. 46(1):322-30). Other such mixtures of disparate splitintein partners have been shown to be unreactive one with another (see,Brenzel, et al., (2006) Biochemistry. 45(6):1571-8). However, it iswithin the ability of a person skilled in the relevant art to determinewhether a particular pair of polypeptides is able to associate with eachother to provide a functional intein, using routine methods and withoutthe exercise of inventive skill.

In some embodiments the IPD101 polypeptide is a circular permutedvariant. In certain embodiments the IPD101 polypeptide is a circularpermuted variant of any one of the polypeptides of SEQ ID NOS: 2, 4, 6,8, 10, 12, 14, 16, 18, 20, 22, 24, 25, 26, 28, 29, 30, 32, 46, 48, 50,52, 54, 56, 58, and 60, or variant thereof having an amino acidsubstitution, deletion, addition or combinations thereof. The approachused in creating new sequences resembles that of naturally occurringpairs of proteins that are related by linear reorganization of theiramino acid sequences (Cunningham, et al., (1979) Proc. Natl. Acad. Sci.U.S.A. 76:3218-3222; Teather and Erfle, (1990) J. Bacteriol.172:3837-3841; Schimming, et al., (1992) Eur. J. Biochem. 204:13-19;Yamiuchi and Minamikawa, (1991) FEBS Lett. 260:127-130; MacGregor, etal., (1996) FEBS Lett. 378:263-266). This type of rearrangement toproteins was described by Goldenberg and Creighton (J. Mol. Biol.165:407-413, 1983). In creating a circular permuted variant a newN-terminus is selected at an internal site (breakpoint) of the originalsequence, the new sequence having the same order of amino acids as theoriginal from the breakpoint until it reaches an amino acid that is ator near the original C-terminus. At this point the new sequence isjoined, either directly or through an additional portion of sequence(linker), to an amino acid that is at or near the original N-terminusand the new sequence continues with the same sequence as the originaluntil it reaches a point that is at or near the amino acid that wasN-terminal to the breakpoint site of the original sequence, this residueforming the new C-terminus of the chain. The length of the amino acidsequence of the linker can be selected empirically or with guidance fromstructural information or by using a combination of the two approaches.When no structural information is available, a small series of linkerscan be prepared for testing using a design whose length is varied inorder to span a range from 0 to 50 Å and whose sequence is chosen inorder to be consistent with surface exposure (hydrophilicity, Hopp andWoods, (1983) Mol. Immunol. 20:483-489; Kyte and Doolittle, (1982) J.Mol. Biol. 157:105-132; solvent exposed surface area, Lee and Richards,(1971) J. Mol. Biol. 55:379-400) and the ability to adopt the necessaryconformation without deranging the configuration of the pesticidalpolypeptide (conformationally flexible; Karplus and Schulz, (1985)Naturwissenschaften 72:212-213). Assuming an average of translation of2.0 to 3.8 Å per residue, this would mean the length to test would bebetween 0 to 30 residues, with 0 to 15 residues being the preferredrange. Exemplary of such an empirical series would be to constructlinkers using a cassette sequence such as Gly-Gly-Gly-Ser repeated ntimes, where n is 1, 2, 3 or 4. Those skilled in the art will recognizethat there are many such sequences that vary in length or compositionthat can serve as linkers with the primary consideration being that theybe neither excessively long nor short (cf., Sandhu, (1992) Critical Rev.Biotech. 12:437-462); if they are too long, entropy effects will likelydestabilize the three-dimensional fold, and may also make foldingkinetically impractical, and if they are too short, they will likelydestabilize the molecule because of torsional or steric strain. Thoseskilled in the analysis of protein structural information will recognizethat using the distance between the chain ends, defined as the distancebetween the c-alpha carbons, can be used to define the length of thesequence to be used or at least to limit the number of possibilitiesthat must be tested in an empirical selection of linkers. They will alsorecognize that it is sometimes the case that the positions of the endsof the polypeptide chain are ill-defined in structural models derivedfrom x-ray diffraction or nuclear magnetic resonance spectroscopy data,and that when true, this situation will therefore need to be taken intoaccount in order to properly estimate the length of the linker required.From those residues whose positions are well defined are selected tworesidues that are close in sequence to the chain ends, and the distancebetween their c-alpha carbons is used to calculate an approximate lengthfor a linker between them. Using the calculated length as a guide,linkers with a range of number of residues (calculated using 2 to 3.8 Åper residue) are then selected. These linkers may be composed of theoriginal sequence, shortened or lengthened as necessary, and whenlengthened the additional residues may be chosen to be flexible andhydrophilic as described above; or optionally the original sequence maybe substituted for using a series of linkers, one example being theGly-Gly-Gly-Ser cassette approach mentioned above; or optionally acombination of the original sequence and new sequence having theappropriate total length may be used.

Sequences of pesticidal polypeptides capable of folding to biologicallyactive states can be prepared by appropriate selection of the beginning(amino terminus) and ending (carboxyl terminus) positions from withinthe original polypeptide chain while using the linker sequence asdescribed above Amino and carboxyl termini are selected from within acommon stretch of sequence, referred to as a breakpoint region, usingthe guidelines described below. A novel amino acid sequence is thusgenerated by selecting amino and carboxyl termini from within the samebreakpoint region. In many cases the selection of the new termini willbe such that the original position of the carboxyl terminus immediatelypreceded that of the amino terminus. However, those skilled in the artwill recognize that selections of termini anywhere within the region mayfunction, and that these will effectively lead to either deletions oradditions to the amino or carboxyl portions of the new sequence. It is acentral tenet of molecular biology that the primary amino acid sequenceof a protein dictates folding to the three-dimensional structurenecessary for expression of its biological function. Methods are knownto those skilled in the art to obtain and interpret three-dimensionalstructural information using x-ray diffraction of single proteinCrystals or nuclear magnetic resonance spectroscopy of proteinsolutions. Examples of structural information that are relevant to theidentification of breakpoint regions include the location and type ofprotein secondary structure (alpha and 3-10 helices, parallel andanti-parallel beta sheets, chain reversals and turns, and loops; Kabschand Sander, (1983) Biopolymers 22:2577-2637); the degree of solventexposure of amino acid residues, the extent and type of interactions ofresidues with one another (Chothia, (1984) Ann. Rev. Biochem.53:537-572) and the static and dynamic distribution of conformationsalong the polypeptide chain (Alber and Mathews, (1987) Methods Enzymol.154:511-533). In some cases additional information is known aboutsolvent exposure of residues; one example is a site ofpost-translational attachment of carbohydrate which is necessarily onthe surface of the protein. When experimental structural information isnot available or is not feasible to obtain, methods are also availableto analyze the primary amino acid sequence in order to make predictionsof protein tertiary and secondary structure, solvent accessibility andthe occurrence of turns and loops. Biochemical methods are alsosometimes applicable for empirically determining surface exposure whendirect structural methods are not feasible; for example, using theidentification of sites of chain scission following limited proteolysisin order to infer surface exposure (Gentile and Salvatore, (1993) Eur.J. Biochem. 218:603-621). Thus using either the experimentally derivedstructural information or predictive methods (e.g., Srinivisan and Rose,(1995) Proteins: Struct., Funct. & Genetics 22:81-99) the parental aminoacid sequence is inspected to classify regions according to whether ornot they are integral to the maintenance of secondary and tertiarystructure. The occurrence of sequences within regions that are known tobe involved in periodic secondary structure (alpha and 3-10 helices,parallel and anti-parallel beta sheets) are regions that should beavoided. Similarly, regions of amino acid sequence that are observed orpredicted to have a low degree of solvent exposure are more likely to bepart of the so-called hydrophobic core of the protein and should also beavoided for selection of amino and carboxyl termini In contrast, thoseregions that are known or predicted to be in surface turns or loops, andespecially those regions that are known not to be required forbiological activity, are the preferred sites for location of theextremes of the polypeptide chain. Continuous stretches of amino acidsequence that are preferred based on the above criteria are referred toas a breakpoint region. Polynucleotides encoding circular permutedIPD101 polypeptides with new N-terminus/C-terminus which contain alinker region separating the original C-terminus and N-terminus can bemade essentially following the method described in Mullins, et al.,(1994) J. Am. Chem. Soc. 116:5529-5533. Multiple steps of polymerasechain reaction (PCR) amplifications are used to rearrange the DNAsequence encoding the primary amino acid sequence of the protein.Polynucleotides encoding circular permuted IPD101 polypeptides with newN-terminus/C-terminus which contain a linker region separating theoriginal C-terminus and N-terminus can be made based on thetandem-duplication method described in Horlick, et al., (1992) ProteinEng. 5:427-431. Polymerase chain reaction (PCR) amplification of the newN-terminus/C-terminus genes is performed using a tandemly duplicatedtemplate DNA.

In another embodiment fusion proteins are provided that include withinits amino acid sequence an amino acid sequence comprising an IPD101polypeptide of the disclosure. Methods for design and construction offusion proteins (and polynucleotides encoding same) are known to thoseof skill in the art. Polynucleotides encoding an IPD101 polypeptide maybe fused to signal sequences which will direct the localization of theIPD101 polypeptide to particular compartments of a prokaryotic oreukaryotic cell and/or direct the secretion of the IPD101 polypeptide ofthe embodiments from a prokaryotic or eukaryotic cell. For example, inE. coli, one may wish to direct the expression of the protein to theperiplasmic space. Examples of signal sequences or proteins (orfragments thereof) to which the IPD101 polypeptide may be fused in orderto direct the expression of the polypeptide to the periplasmic space ofbacteria include, but are not limited to, the pelB signal sequence, themaltose binding protein (MBP) signal sequence, MBP, the ompA signalsequence, the signal sequence of the periplasmic E. coli heat-labileenterotoxin B-subunit and the signal sequence of alkaline phosphatase.Several vectors are commercially available for the construction offusion proteins which will direct the localization of a protein, such asthe pMAL series of vectors (particularly the pMAL-p series) availablefrom New England Biolabs. In a specific embodiment, the IPD101polypeptide may be fused to the pelB pectate lyase signal sequence toincrease the efficiency of expression and purification of suchpolypeptides in Gram-negative bacteria (see, U.S. Pat. Nos. 5,576,195and 5,846,818). Plant plastid transit peptide/polypeptide fusions arewell known in the art. Apoplast transit peptides such as rice or barleyalpha-amylase secretion signal are also well known in the art. Theplastid transit peptide is generally fused N-terminal to the polypeptideto be targeted (e.g., the fusion partner). In one embodiment, the fusionprotein consists essentially of the plastid transit peptide and theIPD101 polypeptide to be targeted. In another embodiment, the fusionprotein comprises the plastid transit peptide and the polypeptide to betargeted. In such embodiments, the plastid transit peptide is preferablyat the N-terminus of the fusion protein. However, additional amino acidresidues may be N-terminal to the plastid transit peptide providing thatthe fusion protein is at least partially targeted to a plastid. In aspecific embodiment, the plastid transit peptide is in the N-terminalhalf, N-terminal third or N-terminal quarter of the fusion protein. Mostor all of the plastid transit peptide is generally cleaved from thefusion protein upon insertion into the plastid. The position of cleavagemay vary slightly between plant species, at different plantdevelopmental stages, as a result of specific intercellular conditionsor the particular combination of transit peptide/fusion partner used. Inone embodiment, the plastid transit peptide cleavage is homogenous suchthat the cleavage site is identical in a population of fusion proteins.In another embodiment, the plastid transit peptide is not homogenous,such that the cleavage site varies by 1-10 amino acids in a populationof fusion proteins. The plastid transit peptide can be recombinantlyfused to a second protein in one of several ways. For example, arestriction endonuclease recognition site can be introduced into thenucleotide sequence of the transit peptide at a position correspondingto its C-terminal end and the same or a compatible site can beengineered into the nucleotide sequence of the protein to be targeted atits N-terminal end. Care must be taken in designing these sites toensure that the coding sequences of the transit peptide and the secondprotein are kept “in frame” to allow the synthesis of the desired fusionprotein. In some cases, it may be preferable to remove the initiatormethionine of the second protein when the new restriction site isintroduced. The introduction of restriction endonuclease recognitionsites on both parent molecules and their subsequent joining throughrecombinant DNA techniques may result in the addition of one or moreextra amino acids between the transit peptide and the second protein.This generally does not affect targeting activity as long as the transitpeptide cleavage site remains accessible and the function of the secondprotein is not altered by the addition of these extra amino acids at itsN-terminus. Alternatively, one skilled in the art can create a precisecleavage site between the transit peptide and the second protein (withor without its initiator methionine) using gene synthesis (Stemmer, etal., (1995) Gene 164:49-53) or similar methods. In addition, the transitpeptide fusion can intentionally include amino acids downstream of thecleavage site. The amino acids at the N-terminus of the mature proteincan affect the ability of the transit peptide to target proteins toplastids and/or the efficiency of cleavage following protein import.This may be dependent on the protein to be targeted. See, e.g., Comai,et al., (1988) J. Biol. Chem. 263(29):15104-9. In some embodiments theIPD101 polypeptide is fused to a heterologous signal peptide orheterologous transit peptide.

In some embodiments fusion proteins are provide comprising an IPD101polypeptide or chimeric IPD101 polypeptide of the disclosure representedby a formula selected from the group consisting of:

R¹-L-R², R²-L-R¹, R¹—R² or R²—R¹

wherein R¹ is an IPD101 polypeptide or chimeric IPD101 polypeptide ofthe disclosure and R² is a protein of interest. In some embodiments R¹and R² are an IPD101 polypeptide or chimeric IPD101 polypeptide of thedisclosure. The R¹ polypeptide is fused either directly or through alinker (L) segment to the R² polypeptide. The term “directly” definesfusions in which the polypeptides are joined without a peptide linker.Thus “L” represents a chemical bound or polypeptide segment to whichboth R¹ and R² are fused in frame, most commonly L is a linear peptideto which R¹ and R² are bound by amide bonds linking the carboxy terminusof R¹ to the amino terminus of L and carboxy terminus of L to the aminoterminus of R². By “fused in frame” is meant that there is notranslation termination or disruption between the reading frames of R¹and R². The linking group (L) is generally a polypeptide of between 1and 500 amino acids in length. The linkers joining the two molecules arepreferably designed to (1) allow the two molecules to fold and actindependently of each other, (2) not have a propensity for developing anordered secondary structure which could interfere with the functionaldomains of the two proteins, (3) have minimal hydrophobic or chargedcharacteristic which could interact with the functional protein domainsand (4) provide steric separation of R¹ and R² such that R¹ and R² couldinteract simultaneously with their corresponding receptors on a singlecell. Typically surface amino acids in flexible protein regions includeGly, Asn and Ser. Virtually any permutation of amino acid sequencescontaining Gly, Asn and Ser would be expected to satisfy the abovecriteria for a linker sequence. Other neutral amino acids, such as Thrand Ala, may also be used in the linker sequence. Additional amino acidsmay also be included in the linkers due to the addition of uniquerestriction sites in the linker sequence to facilitate construction ofthe fusions.

In some embodiments the linkers comprise sequences selected from thegroup of formulas: (Gly₃Ser)_(n), (Gly₄Ser)_(n), (Gly₅Ser)_(n),(Gly_(n)Ser)_(n) or (AlaGlySer)_(n) where n is an integer. One exampleof a highly-flexible linker is the (GlySer)-rich spacer region presentwithin the pIII protein of the filamentous bacteriophages, e.g.bacteriophages M13 or fd (Schaller, et al., 1975). This region providesa long, flexible spacer region between two domains of the pIII surfaceprotein. Also included are linkers in which an endopeptidase recognitionsequence is included. Such a cleavage site may be valuable to separatethe individual components of the fusion to determine if they areproperly folded and active in vitro. Examples of various endopeptidasesinclude, but are not limited to, Plasmin, Enterokinase, Kallikerin,Urokinase, Tissue Plasminogen activator, clostripain, Chymosin,Collagenase, Russell's Viper Venom Protease, Postproline cleavageenzyme, V8 protease, Thrombin and factor Xa. In some embodiments thelinker comprises the amino acids EEKKN (SEQ ID NO:61) from themulti-gene expression vehicle (MGEV), which is cleaved by vacuolarproteases as disclosed in US Patent Application Publication Number US2007/0277263. In other embodiments, peptide linker segments from thehinge region of heavy chain immunoglobulins IgG, IgA, IgM, IgD or IgEprovide an angular relationship between the attached polypeptides.Especially useful are those hinge regions where the cysteines arereplaced with serines. Linkers of the present disclosure includesequences derived from murine IgG gamma 2b hinge region in which thecysteines have been changed to serines. The fusion proteins are notlimited by the form, size or number of linker sequences employed and theonly requirement of the linker is that functionally it does notinterfere adversely with the folding and function of the individualmolecules of the fusion.

Nucleic Acid Molecules, and Variants and Fragments Thereof

Isolated or recombinant nucleic acid molecules comprising nucleic acidsequences encoding IPD101 polypeptides or biologically active portionsthereof, as well as nucleic acid molecules sufficient for use ashybridization probes to identify nucleic acid molecules encodingproteins with regions of sequence homology are provided. As used herein,the term “nucleic acid molecule” refers to DNA molecules (e.g.,recombinant DNA, cDNA, genomic DNA, plastid DNA, mitochondrial DNA) andRNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated usingnucleotide analogs. The nucleic acid molecule can be single-stranded ordouble-stranded, but preferably is double-stranded DNA.

An “isolated” nucleic acid molecule (or DNA) is used herein to refer toa nucleic acid sequence (or DNA) that is no longer in its naturalenvironment, for example in vitro. A “recombinant” nucleic acid molecule(or DNA) is used herein to refer to a nucleic acid sequence (or DNA)that is in a recombinant bacterial or plant host cell. In someembodiments, an “isolated” or “recombinant” nucleic acid is free ofsequences (preferably protein encoding sequences) that naturally flankthe nucleic acid (i.e., sequences located at the 5′ and 3′ ends of thenucleic acid) in the genomic DNA of the organism from which the nucleicacid is derived. For purposes of the disclosure, “isolated” or“recombinant” when used to refer to nucleic acid molecules excludesisolated chromosomes. For example, in various embodiments, therecombinant nucleic acid molecules encoding IPD101 polypeptides cancontain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kbof nucleic acid sequences that naturally flank the nucleic acid moleculein genomic DNA of the cell from which the nucleic acid is derived.

In some embodiments an isolated nucleic acid molecule encoding IPD101polypeptides has one or more change in the nucleic acid sequencecompared to the native or genomic nucleic acid sequence. In someembodiments the change in the native or genomic nucleic acid sequenceincludes but is not limited to: changes in the nucleic acid sequence dueto the degeneracy of the genetic code; changes in the nucleic acidsequence due to the amino acid substitution, insertion, deletion and/oraddition compared to the native or genomic sequence; removal of one ormore intron; deletion of one or more upstream or downstream regulatoryregions; and deletion of the 5′ and/or 3′ untranslated region associatedwith the genomic nucleic acid sequence. In some embodiments the nucleicacid molecule encoding an IPD101 polypeptide is a non-genomic sequence.

A variety of polynucleotides that encode IPD101 polypeptides or relatedproteins are contemplated. Such polynucleotides are useful forproduction of IPD101 polypeptides in host cells when operably linked toa suitable promoter, transcription termination and/or polyadenylationsequences. Such polynucleotides are also useful as probes for isolatinghomologous or substantially homologous polynucleotides that encodeIPD101 polypeptides or related proteins.

Polynucleotides Encoding IPD101 Polypeptides

One source of polynucleotides that encode IPD101 polypeptides or relatedproteins is a Lysinibacillus bacterium which may contain an IPD101polynucleotide of any one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 19,21, or 23, encoding an IPD101 polypeptide of SEQ ID NOs: 2, 4, 6, 8, 10,12, 14, 16, 20, 22, or 24, respectively. The polynucleotides of any oneor more of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 27,31, 45, 47, 49, 51, 53, 55, 57, or 59, can be used to express IPD101polypeptides in recombinant bacterial hosts that include but are notlimited to Agrobacterium, Bacillus, Escherichia, Salmonella,Lysinibacillus, Acetobacter, Pseudomonas and Rhizobium bacterial hostcells. The polynucleotides are also useful as probes for isolatinghomologous or substantially homologous polynucleotides encoding IPD101polypeptides or related proteins. Such probes can be used to identifyhomologous or substantially homologous polynucleotides derived fromPseudomonas species.

Polynucleotides encoding IPD101 polypeptides can also be synthesized denovo from an IPD101 polypeptide sequence. The sequence of thepolynucleotide gene can be deduced from an IPD101 polypeptide sequencethrough use of the genetic code. Computer programs such as“BackTranslate” (GCG™ Package, Acclerys, Inc. San Diego, Calif.) can beused to convert a peptide sequence to the corresponding nucleotidesequence encoding the peptide. Examples of IPD101 polypeptide sequencesthat can be used to obtain corresponding nucleotide encoding sequencesinclude, but are not limited to the IPD101 polypeptides of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 25, 26, 28, 29, 30, 32, 46,48, 50, 52, 54, 56, 58, and 60. Furthermore, synthetic IPD101polynucleotide sequences of the disclosure can be designed so that theywill be expressed in plants.

In some embodiments the nucleic acid molecule encoding an IPD101polypeptide is a polynucleotide having the sequence set forth in any oneof SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 27, 31, 45,47, 49, 51, 53, 55, 57, or 59, and variants, fragments and complementsthereof. “Complement” is used herein to refer to a nucleic acid sequencethat is sufficiently complementary to a given nucleic acid sequence suchthat it can hybridize to the given nucleic acid sequence to thereby forma stable duplex. “Polynucleotide sequence variants” is used herein torefer to a nucleic acid sequence that except for the degeneracy of thegenetic code encodes the same polypeptide.

In some embodiments the nucleic acid molecule encoding the IPD101polypeptide is a non-genomic nucleic acid sequence. As used herein a“non-genomic nucleic acid sequence” or “non-genomic nucleic acidmolecule” or “non-genomic polynucleotide” refers to a nucleic acidmolecule that has one or more change in the nucleic acid sequencecompared to a native or genomic nucleic acid sequence. In someembodiments the change to a native or genomic nucleic acid moleculeincludes but is not limited to: changes in the nucleic acid sequence dueto the degeneracy of the genetic code; optimization of the nucleic acidsequence for expression in plants; changes in the nucleic acid sequenceto introduce at least one amino acid substitution, insertion, deletionand/or addition compared to the native or genomic sequence; removal ofone or more intron associated with the genomic nucleic acid sequence;insertion of one or more heterologous introns; deletion of one or moreupstream or downstream regulatory regions associated with the genomicnucleic acid sequence; insertion of one or more heterologous upstream ordownstream regulatory regions; deletion of the 5′ and/or 3′ untranslatedregion associated with the genomic nucleic acid sequence; insertion of aheterologous 5′ and/or 3′ untranslated region; and modification of apolyadenylation site. In some embodiments the non-genomic nucleic acidmolecule is a synthetic nucleic acid sequence.

In some embodiments the nucleic acid molecule encoding an IPD101polypeptide disclosed herein is a non-genomic polynucleotide having anucleotide sequence having at least 50%, 51%, 52%, 53%, 54%, 55%, 56%,57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%,71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or greater identity, to the nucleic acid sequence of any one of SEQID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 27, 31, 45, 47, 49,51, 53, 55, 57, or 59, wherein the IPD101 polypeptide has insecticidalactivity.

In some embodiments the nucleic acid molecule encodes an IPD101polypeptide variant comprising one or more amino acid substitutions tothe amino acid sequence of any one of SEQ ID NOS: 2, 4, 6, 8, 10, 12,14, 16, 18, 20, 22, 24, 25, 26, 28, 29, 30, 32, 46, 48, 50, 52, 54, 56,58, and 60.

Also provided are nucleic acid molecules that encode transcriptionand/or translation products that are subsequently spliced to ultimatelyproduce functional IPD101 polypeptides. Splicing can be accomplished invitro or in vivo, and can involve cis- or trans-splicing. The substratefor splicing can be polynucleotides (e.g., RNA transcripts) orpolypeptides. An example of cis-splicing of a polynucleotide is where anintron inserted into a coding sequence is removed and the two flankingexon regions are spliced to generate an IPD101 polypeptide encodingsequence. An example of trans-splicing would be where a polynucleotideis encrypted by separating the coding sequence into two or morefragments that can be separately transcribed and then spliced to formthe full-length pesticidal encoding sequence. The use of a splicingenhancer sequence, which can be introduced into a construct, canfacilitate splicing either in cis or trans-splicing of polypeptides(U.S. Pat. Nos. 6,365,377 and 6,531,316). Thus, in some embodiments thepolynucleotides do not directly encode a full-length IPD101 polypeptide,but rather encode a fragment or fragments of an IPD101 polypeptide.These polynucleotides can be used to express a functional IPD101polypeptide through a mechanism involving splicing, where splicing canoccur at the level of polynucleotide (e.g., intron/exon) and/orpolypeptide (e.g., intein/extein). This can be useful, for example, incontrolling expression of pesticidal activity, since a functionalpesticidal polypeptide will only be expressed if all required fragmentsare expressed in an environment that permits splicing processes togenerate functional product. In another example, introduction of one ormore insertion sequences into a polynucleotide can facilitaterecombination with a low homology polynucleotide; use of an intron orintein for the insertion sequence facilitates the removal of theintervening sequence, thereby restoring function of the encoded variant.

Nucleic acid molecules that are fragments of these nucleic acidsequences encoding IPD101 polypeptides are also encompassed by theembodiments. “Fragment” as used herein refers to a portion of thenucleic acid sequence encoding an IPD101 polypeptide. A fragment of anucleic acid sequence may encode a biologically active portion of anIPD101 polypeptide or it may be a fragment that can be used as ahybridization probe or PCR primer using methods disclosed below. Nucleicacid molecules that are fragments of a nucleic acid sequence encoding anIPD101 polypeptide comprise at least about 150, 180, 210, 240, 270, 300,330, 360, 400, 450, or 500 contiguous nucleotides or up to the number ofnucleotides present in a full-length nucleic acid sequence encoding anIPD101 polypeptide disclosed herein, depending upon the intended use.“Contiguous nucleotides” is used herein to refer to nucleotide residuesthat are immediately adjacent to one another. Fragments of the nucleicacid sequences of the embodiments will encode protein fragments thatretain the biological activity of the IPD101 polypeptide and, hence,retain insecticidal activity. “Retains insecticidal activity” is usedherein to refer to a polypeptide having at least about 10%, at leastabout 30%, at least about 50%, at least about 70%, 80%, 90%, 95% orhigher of the insecticidal activity of any one of the full-length IPD101polypeptides set forth in SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18,20, 22, 24, 25, 26, 28, 29, 30, 32, 46, 48, 50, 52, 54, 56, 58, and 60.In some embodiments, the insecticidal activity is against a Lepidopteranspecies. In one embodiment, the insecticidal activity is against aColeopteran species. In some embodiments, the insecticidal activity isagainst one or more insect pests of the corn rootworm complex: westerncorn rootworm, Diabrotica virgifera; northern corn rootworm, D. barberi:Southern corn rootworm or spotted cucumber beetle; Diabroticaundecimpunctata howardi, Diabrotica speciosa, and the Mexican cornrootworm, D. virgifera zeae. In one embodiment, the insecticidalactivity is against a Diabrotica species.

In some embodiments the IPD101 polypeptide is encoded by a nucleic acidsequence sufficiently homologous to any one of the nucleic acidsequences of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 27,31, 45, 47, 49, 51, 53, 55, 57, or 59.

To determine the percent identity of two amino acid sequences or of twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes. The percent identity between the two sequences is a functionof the number of identical positions shared by the sequences (i.e.,percent identity=number of identical positions/total number of positions(e.g., overlapping positions)×100). In one embodiment, the two sequencesare the same length. In another embodiment, the comparison is across theentirety of the reference sequence (e.g., across the entirety of SEQ IDNO: 1). The percent identity between two sequences can be determinedusing techniques similar to those described below, with or withoutallowing gaps. In calculating percent identity, typically exact matchesare counted.

Another non-limiting example of a mathematical algorithm utilized forthe comparison of sequences is the algorithm of Needleman and Wunsch,(1970) J. Mol. Biol. 48(3):443-453, used GAP Version 10 software todetermine sequence identity or similarity using the following defaultparameters: % identity and % similarity for a nucleic acid sequenceusing GAP Weight of 50 and Length Weight of 3, and the nwsgapdna.cmpiiscoring matrix; % identity or % similarity for an amino acid sequenceusing GAP weight of 8 and length weight of 2, and the BLOSUM62 scoringprogram. Equivalent programs may also be used. “Equivalent program” isused herein to refer to any sequence comparison program that, for anytwo sequences in question, generates an alignment having identicalnucleotide residue matches and an identical percent sequence identitywhen compared to the corresponding alignment generated by GAP Version10.

In some embodiments an IPD101 polynucleotide encodes an IPD101polypeptide comprising an amino acid sequence having at least about 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or greater identity across the entire length ofthe amino acid sequence of any one of SEQ ID NOS: 2, 4, 6, 8, 10, 12,14, 16, 18, 20, 22, 24, 25, 26, 28, 29, 30, 32, 46, 48, 50, 52, 54, 56,58, and 60.

In some embodiments polynucleotides are provided encoding chimericpolypeptides comprising regions of at least two different IPD101polypeptides of the disclosure.

In some embodiments polynucleotides are provided encoding chimericpolypeptides comprising an N-terminal Region of a first IPD101polypeptide of the disclosure operably fused to a C-terminal Region of asecond IPD101 polypeptide of the disclosure.

The embodiments also encompass nucleic acid molecules encoding IPD101polypeptide variants. “Variants” of the IPD101 polypeptide encodingnucleic acid sequences include those sequences that encode the IPD101polypeptides disclosed herein but that differ conservatively because ofthe degeneracy of the genetic code as well as those that aresufficiently identical as discussed above. Naturally occurring allelicvariants can be identified with the use of well-known molecular biologytechniques, such as polymerase chain reaction (PCR) and hybridizationtechniques as outlined below. Variant nucleic acid sequences alsoinclude synthetically derived nucleic acid sequences that have beengenerated, for example, by using site-directed mutagenesis but whichstill encode the IPD101 polypeptides disclosed as discussed below.

The present disclosure provides isolated or recombinant polynucleotidesthat encode any of the IPD101 polypeptides disclosed herein. Thosehaving ordinary skill in the art will readily appreciate that due to thedegeneracy of the genetic code, a multitude of nucleotide sequencesencoding IPD101 polypeptides of the present disclosure exist.

The skilled artisan will further appreciate that changes can beintroduced by mutation of the nucleic acid sequences thereby leading tochanges in the amino acid sequence of the encoded IPD101 polypeptides,without altering the biological activity of the proteins. Thus, variantnucleic acid molecules can be created by introducing one or morenucleotide substitutions, additions and/or deletions into thecorresponding nucleic acid sequence disclosed herein, such that one ormore amino acid substitutions, additions or deletions are introducedinto the encoded protein. Mutations can be introduced by standardtechniques, such as site-directed mutagenesis and PCR-mediatedmutagenesis. Such variant nucleic acid sequences are also encompassed bythe present disclosure.

Alternatively, variant nucleic acid sequences can be made by introducingmutations randomly along all or part of the coding sequence, such as bysaturation mutagenesis, and the resultant mutants can be screened forability to confer pesticidal activity to identify mutants that retainactivity. Following mutagenesis, the encoded protein can be expressedrecombinantly, and the activity of the protein can be determined usingstandard assay techniques.

The polynucleotides of the disclosure and fragments thereof areoptionally used as substrates for a variety of recombination andrecursive recombination reactions, in addition to standard cloningmethods as set forth in, e.g., Ausubel, Berger and Sambrook, i.e., toproduce additional pesticidal polypeptide homologues and fragmentsthereof with desired properties. A variety of such reactions are known.Methods for producing a variant of any nucleic acid listed hereincomprising recursively recombining such polynucleotide with a second (ormore) polynucleotide, thus forming a library of variant polynucleotidesare also embodiments of the disclosure, as are the libraries produced,the cells comprising the libraries and any recombinant polynucleotideproduced by such methods. Additionally, such methods optionally compriseselecting a variant polynucleotide from such libraries based onpesticidal activity, as is wherein such recursive recombination is donein vitro or in vivo.

A variety of diversity generating protocols, including nucleic acidrecursive recombination protocols are available and fully described inthe art. The procedures can be used separately, and/or in combination toproduce one or more variants of a nucleic acid or set of nucleic acids,as well as variants of encoded proteins. Individually and collectively,these procedures provide robust, widely applicable ways of generatingdiversified nucleic acids and sets of nucleic acids (including, e.g.,nucleic acid libraries) useful, e.g., for the engineering or rapidevolution of nucleic acids, proteins, pathways, cells and/or organismswith new and/or improved characteristics.

While distinctions and classifications are made in the course of theensuing discussion for clarity, it will be appreciated that thetechniques are often not mutually exclusive. Indeed, the various methodscan be used singly or in combination, in parallel or in series, toaccess diverse sequence variants.

The result of any of the diversity generating procedures describedherein can be the generation of one or more nucleic acids, which can beselected or screened for nucleic acids with or which confer desirableproperties or that encode proteins with or which confer desirableproperties. Following diversification by one or more of the methodsherein or otherwise available to one of skill, any nucleic acids thatare produced can be selected for a desired activity or property, e.g.pesticidal activity or, such activity at a desired pH, etc. This caninclude identifying any activity that can be detected, for example, inan automated or automatable format, by any of the assays in the art,see, e.g., discussion of screening of insecticidal activity, infra. Avariety of related (or even unrelated) properties can be evaluated, inserial or in parallel, at the discretion of the practitioner.

Descriptions of a variety of diversity generating procedures forgenerating modified nucleic acid sequences, e.g., those coding forpolypeptides having pesticidal activity or fragments thereof, are foundin the following publications and the references cited therein: Soong,et al., (2000) Nat Genet 25(4):436-439; Stemmer, et al., (1999) TumorTargeting 4:1-4; Ness, et al., (1999) Nat Biotechnol 17:893-896; Chang,et al., (1999) Nat Biotechnol 17:793-797; Minshull and Stemmer, (1999)Curr Opin Chem Biol 3:284-290; Christians, et al., (1999) Nat Biotechnol17:259-264; Crameri, et al., (1998) Nature 391:288-291; Crameri, et al.,(1997) Nat Biotechnol 15:436-438; Zhang, et al., (1997) PNAS USA94:4504-4509; Patten, et al., (1997) Curr Opin Biotechnol 8:724-733;Crameri, et al., (1996) Nat Med 2:100-103; Crameri, et al., (1996) NatBiotechnol 14:315-319; Gates, et al., (1996) J Mol Biol 255:373-386;Stemmer, (1996) “Sexual PCR and Assembly PCR” In: The Encyclopedia ofMolecular Biology. VCH Publishers, New York. pp. 447-457; Crameri andStemmer, (1995) BioTechniques 18:194-195; Stemmer, et al., (1995) Gene,164:49-53; Stemmer, (1995) Science 270: 1510; Stemmer, (1995)Bio/Technology 13:549-553; Stemmer, (1994) Nature 370:389-391 andStemmer, (1994) PNAS USA 91:10747-10751.

Mutational methods of generating diversity include, for example,site-directed mutagenesis (Ling, et al., (1997) Anal Biochem254(2):157-178; Dale, et al., (1996) Methods Mol Biol 57:369-374; Smith,(1985) Ann Rev Genet 19:423-462; Botstein and Shortle, (1985) Science229:1193-1201; Carter, (1986) Biochem J 237:1-7 and Kunkel, (1987) “Theefficiency of oligonucleotide directed mutagenesis” in Nucleic Acids &Molecular Biology (Eckstein and Lilley, eds., Springer Verlag, Berlin));mutagenesis using uracil containing templates (Kunkel, (1985) PNAS USA82:488-492; Kunkel, et al., (1987) Methods Enzymol 154:367-382 and Bass,et al., (1988) Science 242:240-245); oligonucleotide-directedmutagenesis (Zoller and Smith, (1983) Methods Enzymol 100:468-500;Zoller and Smith, (1987) Methods Enzymol 154:329-350 (1987); Zoller andSmith, (1982) Nucleic Acids Res 10:6487-6500), phosphorothioate-modifiedDNA mutagenesis (Taylor, et al., (1985) Nucl Acids Res 13:8749-8764;Taylor, et al., (1985) Nucl Acids Res 13:8765-8787 (1985); Nakamaye andEckstein, (1986) Nucl Acids Res 14:9679-9698; Sayers, et al., (1988)Nucl Acids Res 16:791-802 and Sayers, et al., (1988) Nucl Acids Res16:803-814); mutagenesis using gapped duplex DNA (Kramer, et al., (1984)Nucl Acids Res 12:9441-9456; Kramer and Fritz, (1987) Methods Enzymol154:350-367; Kramer, et al., (1988) Nucl Acids Res 16:7207 and Fritz, etal., (1988) Nucl Acids Res 16:6987-6999).

Additional suitable methods include point mismatch repair (Kramer, etal., (1984) Cell 38:879-887), mutagenesis using repair-deficient hoststrains (Carter, et al., (1985) Nucl Acids Res 13:4431-4443 and Carter,(1987) Methods in Enzymol 154:382-403), deletion mutagenesis(Eghtedarzadeh and Henikoff, (1986) Nucl Acids Res 14:5115),restriction-selection and restriction-purification (Wells, et al.,(1986) Phil Trans R Soc Lond A 317:415-423), mutagenesis by total genesynthesis (Nambiar, et al., (1984) Science 223:1299-1301; Sakamar andKhorana, (1988) Nucl Acids Res 14:6361-6372; Wells, et al., (1985) Gene34:315-323 and Grundström, et al., (1985) Nucl Acids Res 13:3305-3316),double-strand break repair (Mandecki, (1986) PNAS USA, 83:7177-7181 andArnold, (1993) Curr Opin Biotech 4:450-455). Additional details on manyof the above methods can be found in Methods Enzymol Volume 154, whichalso describes useful controls for trouble-shooting problems withvarious mutagenesis methods.

Additional details regarding various diversity generating methods can befound in the following US patents, PCT Publications and Applications andEPO publications: U.S. Pat. Nos. 5,723,323, 5,763,192, 5,814,476,5,817,483, 5,824,514, 5,976,862, 5,605,793, 5,811,238, 5,830,721,5,834,252, 5,837,458, WO 1995/22625, WO 1996/33207, WO 1997/20078, WO1997/35966, WO 1999/41402, WO 1999/41383, WO 1999/41369, WO 1999/41368,EP 752008, EP 1012670, WO 1999/23107, WO 1999/21979, WO 1998/31837, WO1998/27230, WO 1998/27230, WO 2000/00632, WO 2000/09679, WO 1998/42832,WO 1999/29902, WO 1998/41653, WO 1998/41622, WO 1998/42727, WO2000/18906, WO 2000/04190, WO 2000/42561, WO 2000/42559, WO 2000/42560,WO 2001/23401 and PCT/US01/06775.

The nucleotide sequences of the embodiments can also be used to isolatecorresponding sequences from a bacterial source, including but notlimited to a Pseudomonas species. In this manner, methods such as PCR,hybridization, and the like can be used to identify such sequences basedon their sequence homology to the sequences set forth herein. Sequencesthat are selected based on their sequence identity to the entiresequences set forth herein or to fragments thereof are encompassed bythe embodiments. Such sequences include sequences that are orthologs ofthe disclosed sequences. The term “orthologs” refers to genes derivedfrom a common ancestral gene and which are found in different species asa result of speciation. Genes found in different species are consideredorthologs when their nucleotide sequences and/or their encoded proteinsequences share substantial identity as defined elsewhere herein.Functions of orthologs are often highly conserved among species.

In a PCR approach, oligonucleotide primers can be designed for use inPCR reactions to amplify corresponding DNA sequences from cDNA orgenomic DNA extracted from any organism of interest. Methods fordesigning PCR primers and PCR cloning are generally known in the art andare disclosed in Sambrook, et al., (1989) Molecular Cloning: ALaboratory Manual (2d ed., Cold Spring Harbor Laboratory Press,Plainview, N.Y.), hereinafter “Sambrook”. See also, Innis, et al., eds.(1990) PCR Protocols: A Guide to Methods and Applications (AcademicPress, New York); Innis and Gelfand, eds. (1995) PCR Strategies(Academic Press, New York); and Innis and Gelfand, eds. (1999) PCRMethods Manual (Academic Press, New York). Known methods of PCR include,but are not limited to, methods using paired primers, nested primers,single specific primers, degenerate primers, gene-specific primers,vector-specific primers, partially-mismatched primers, and the like.

To identify potential IPD101 polypeptides from bacterium collections,the bacterial cell lysates can be screened with antibodies generatedagainst IPD101 polypeptides using Western blotting and/or ELISA methods.This type of assay can be performed in a high throughput fashion.Positive samples can be further analyzed by various techniques such asantibody based protein purification and identification. Methods ofgenerating antibodies are well known in the art as discussed infra.

Alternatively, mass spectrometry based protein identification method canbe used to identify homologs of IPD101 polypeptides using protocols inthe literatures (Scott Patterson, (1998), 10.22, 1-24, Current Protocolin Molecular Biology published by John Wiley & Son Inc). Specifically,LC-MS/MS based protein identification method is used to associate the MSdata of given cell lysate or desired molecular weight enriched samples(excised from SDS-PAGE gel of relevant molecular weight bands to IPD101polypeptides) with sequence information of an IPD101 polypeptidedisclosed herein. Any match in peptide sequences indicates the potentialof having the homologous proteins in the samples. Additional techniques(protein purification and molecular biology) can be used to isolate theprotein and identify the sequences of the homologs.

In hybridization methods, all or part of the pesticidal nucleic acidsequence can be used to screen cDNA or genomic libraries. Methods forconstruction of such cDNA and genomic libraries are generally known inthe art and are disclosed in Sambrook and Russell, (2001), supra. Theso-called hybridization probes may be genomic DNA fragments, cDNAfragments, RNA fragments or other oligonucleotides and may be labeledwith a detectable group such as 32P or any other detectable marker, suchas other radioisotopes, a fluorescent compound, an enzyme or an enzymeco-factor. Probes for hybridization can be made by labeling syntheticoligonucleotides based on the known IPD101 polypeptide-encoding nucleicacid sequences disclosed herein. Degenerate primers designed on thebasis of conserved nucleotides or amino acid residues in the nucleicacid sequence or encoded amino acid sequence can additionally be used.The probe typically comprises a region of nucleic acid sequence thathybridizes under stringent conditions to at least about 12, at leastabout 25, at least about 50, 75, 100, 125, 150, 175 or 200 consecutivenucleotides of nucleic acid sequences encoding IPD101 polypeptides ofthe disclosure or a fragment or variant thereof. Methods for thepreparation of probes for hybridization and stringency conditions aregenerally known in the art and are disclosed in Sambrook and Russell,(2001), supra, herein incorporated by reference.

For example, an entire nucleic acid sequence, encoding an IPD101polypeptide, disclosed herein or one or more portions thereof may beused as a probe capable of specifically hybridizing to correspondingnucleic acid sequences encoding IPD101 polypeptide-like sequences andmessenger RNAs. To achieve specific hybridization under a variety ofconditions, such probes include sequences that are unique and arepreferably at least about 10 nucleotides in length or at least about 20nucleotides in length. Such probes may be used to amplify correspondingpesticidal sequences from a chosen organism by PCR. This technique maybe used to isolate additional coding sequences from a desired organismor as a diagnostic assay to determine the presence of coding sequencesin an organism. Hybridization techniques include hybridization screeningof plated DNA libraries (either plaques or colonies; see, for example,Sambrook, et al., (1989) Molecular Cloning: A Laboratory Manual (2d ed.,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).

Hybridization of such sequences may be carried out under stringentconditions. “Stringent conditions” or “stringent hybridizationconditions” is used herein to refer to conditions under which a probewill hybridize to its target sequence to a detectably greater degreethan to other sequences (e.g., at least 2-fold over background).Stringent conditions are sequence-dependent and will be different indifferent circumstances. By controlling the stringency of thehybridization and/or washing conditions, target sequences that are 100%complementary to the probe can be identified (homologous probing).Alternatively, stringency conditions can be adjusted to allow somemismatching in sequences so that lower degrees of similarity aredetected (heterologous probing). Generally, a probe is less than about1000 nucleotides in length, preferably less than 500 nucleotides inlength

Antibodies

Antibodies to an IPD101 polypeptide of the embodiments or to variants orfragments thereof are also encompassed. The antibodies of the disclosureinclude polyclonal and monoclonal antibodies as well as fragmentsthereof which retain their ability to bind to an IPD101 polypeptide. Anantibody, monoclonal antibody or fragment thereof is said to be capableof binding a molecule if it is capable of specifically reacting with themolecule to thereby bind the molecule to the antibody, monoclonalantibody or fragment thereof. The term “antibody” (Ab) or “monoclonalantibody” (Mab) is meant to include intact molecules as well asfragments or binding regions or domains thereof (such as, for example,Fab and F(ab).sub.2 fragments) which are capable of binding hapten. Suchfragments are typically produced by proteolytic cleavage, such as papainor pepsin. Alternatively, hapten-binding fragments can be producedthrough the application of recombinant DNA technology or throughsynthetic chemistry. Methods for the preparation of the antibodies ofthe present disclosure are generally known in the art. For example, see,Antibodies, A Laboratory Manual, Ed Harlow and David Lane (eds.) ColdSpring Harbor Laboratory, N.Y. (1988), as well as the references citedtherein. Standard reference works setting forth the general principlesof immunology include: Klein, J. Immunology: The Science of Cell-NoncellDiscrimination, John Wiley & Sons, N.Y. (1982); Dennett, et al.,Monoclonal Antibodies, Hybridoma: A New Dimension in BiologicalAnalyses, Plenum Press, N.Y. (1980) and Campbell, “Monoclonal AntibodyTechnology,” In Laboratory Techniques in Biochemistry and MolecularBiology, Vol. 13, Burdon, et al., (eds.), Elsevier, Amsterdam (1984).See also, U.S. Pat. Nos. 4,196,265; 4,609,893; 4,713,325; 4,714,681;4,716,111; 4,716,117 and 4,720,459. Antibodies against IPD101polypeptides or antigen-binding portions thereof can be produced by avariety of techniques, including conventional monoclonal antibodymethodology, for example the standard somatic cell hybridizationtechnique of Kohler and Milstein, (1975) Nature 256:495. Othertechniques for producing monoclonal antibody can also be employed suchas viral or oncogenic transformation of B lymphocytes. An animal systemfor preparing hybridomas is a murine system. Immunization protocols andtechniques for isolation of immunized splenocytes for fusion are knownin the art. Fusion partners (e.g., murine myeloma cells) and fusionprocedures are also known. The antibody and monoclonal antibodies of thedisclosure can be prepared by utilizing an IPD101 polypeptide asantigens. A kit for detecting the presence of an IPD101 polypeptide ordetecting the presence of a nucleotide sequence encoding an IPD101polypeptide in a sample is provided. In one embodiment, the kit providesantibody-based reagents for detecting the presence of an IPD101polypeptide in a tissue sample. In another embodiment, the kit provideslabeled nucleic acid probes useful for detecting the presence of one ormore polynucleotides encoding an IPD101 polypeptide. The kit is providedalong with appropriate reagents and controls for carrying out adetection method, as well as instructions for use of the kit.

Receptor Identification and Isolation

Receptors to the IPD101 polypeptides of the embodiments or to variantsor fragments thereof are also encompassed. Methods for identifyingreceptors are well known in the art (see, Hofmann, et. al., (1988) Eur.J. Biochem. 173:85-91; Gill, et al., (1995) J. Biol. Chem. 27277-27282)can be employed to identify and isolate the receptor that recognizes theIPD101 polypeptide using the brush-border membrane vesicles fromsusceptible insects. In addition to the radioactive labeling methodlisted in the cited literatures, an IPD101 polypeptide can be labeledwith fluorescent dye and other common labels such as streptavidin.Brush-border membrane vesicles (BBMV) of susceptible insects such assoybean looper and stink bugs can be prepared according to the protocolslisted in the references of Hofmann and Gill above and separated onSDS-PAGE gel and blotted on suitable membrane. Labeled IPD101polypeptide can be incubated with blotted membrane of BBMV and labeledIPD101 polypeptide can be identified with the labeled reporters.Identification of protein band(s) that interact with the IPD101polypeptide can be detected by N-terminal amino acid gas phasesequencing or mass spectrometry based protein identification method(Patterson, (1998) 10.22, 1-24, Current Protocol in Molecular Biologypublished by John Wiley & Son Inc). Once the protein is identified, thecorresponding gene can be cloned from genomic DNA or cDNA library of thesusceptible insects and binding affinity can be measured directly withthe IPD101 polypeptide. Receptor function for insecticidal activity bythe IPD101 polypeptide can be verified by RNAi type of gene knock outmethod (Rajagopal, et al., (2002) J. Biol. Chem. 277:46849-46851).

Nucleotide Constructs, Expression Cassettes and Vectors

The use of the term “nucleotide constructs” herein is not intended tolimit the embodiments to nucleotide constructs comprising DNA. Those ofordinary skill in the art will recognize that nucleotide constructs,particularly polynucleotides and oligonucleotides composed ofribonucleotides and combinations of ribonucleotides anddeoxyribonucleotides, may also be employed in the methods disclosedherein. The nucleotide constructs, nucleic acids, and nucleotidesequences of the embodiments additionally encompass all complementaryforms of such constructs, molecules, and sequences. Further, thenucleotide constructs, nucleotide molecules, and nucleotide sequences ofthe embodiments encompass all nucleotide constructs, molecules, andsequences which can be employed in the methods of the embodiments fortransforming plants including, but not limited to, those comprised ofdeoxyribonucleotides, ribonucleotides, and combinations thereof. Suchdeoxyribonucleotides and ribonucleotides include both naturallyoccurring molecules and synthetic analogues. The nucleotide constructs,nucleic acids, and nucleotide sequences of the embodiments alsoencompass all forms of nucleotide constructs including, but not limitedto, single-stranded forms, double-stranded forms, hairpins,stem-and-loop structures and the like.

A further embodiment relates to a transformed organism such as anorganism selected from plant and insect cells, bacteria, yeast,baculovirus, protozoa, nematodes and algae. The transformed organismcomprises a DNA molecule of the embodiments, an expression cassettecomprising the DNA molecule or a vector comprising the expressioncassette, which may be stably incorporated into the genome of thetransformed organism.

The sequences of the embodiments are provided in DNA constructs forexpression in the organism of interest. The construct will include 5′and 3′ regulatory sequences operably linked to a sequence of theembodiments. The term “operably linked” as used herein refers to afunctional linkage between a promoter and a second sequence, wherein thepromoter sequence initiates and mediates transcription of the DNAsequence corresponding to the second sequence. Generally, operablylinked means that the nucleic acid sequences being linked are contiguousand where necessary to join two protein coding regions in the samereading frame. The construct may additionally contain at least oneadditional gene to be cotransformed into the organism. Alternatively,the additional gene(s) can be provided on multiple DNA constructs.

Such a DNA construct is provided with a plurality of restriction sitesfor insertion of the IPD101 polypeptide gene sequence of the disclosureto be under the transcriptional regulation of the regulatory regions.The DNA construct may additionally contain selectable marker genes.

The DNA construct will generally include in the 5′ to 3′ direction oftranscription: a transcriptional and translational initiation region(i.e., a promoter), a DNA sequence of the embodiments, and atranscriptional and translational termination region (i.e., terminationregion) functional in the organism serving as a host. Thetranscriptional initiation region (i.e., the promoter) may be native,analogous, foreign or heterologous to the host organism and/or to thesequence of the embodiments. Additionally, the promoter may be thenatural sequence or alternatively a synthetic sequence. The term“foreign” as used herein indicates that the promoter is not found in thenative organism into which the promoter is introduced. Where thepromoter is “foreign” or “heterologous” to the sequence of theembodiments, it is intended that the promoter is not the native ornaturally occurring promoter for the operably linked sequence of theembodiments. As used herein, a chimeric gene comprises a coding sequenceoperably linked to a transcription initiation region that isheterologous to the coding sequence. Where the promoter is a native ornatural sequence, the expression of the operably linked sequence isaltered from the wild-type expression, which results in an alteration inphenotype.

In some embodiments the DNA construct comprises a polynucleotideencoding an IPD101 polypeptide of the embodiments. In some embodimentsthe DNA construct comprises a polynucleotide encoding a fusion proteincomprising an IPD101 polypeptide of the embodiments.

In some embodiments the DNA construct may also include a transcriptionalenhancer sequence. As used herein, the term an “enhancer” refers to aDNA sequence which can stimulate promoter activity, and may be an innateelement of the promoter or a heterologous element inserted to enhancethe level or tissue-specificity of a promoter. Various enhancers areknown in the art including for example, introns with gene expressionenhancing properties in plants (US Patent Application Publication Number2009/0144863, the ubiquitin intron (i.e., the maize ubiquitin intron 1(see, for example, NCBI sequence S94464)), the omega enhancer or theomega prime enhancer (Gallie, et al., (1989) Molecular Biology of RNAed. Cech (Liss, New York) 237-256 and Gallie, et al., (1987) Gene60:217-25), the CaMV 35S enhancer (see, e.g., Benfey, et al., (1990)EMBO J. 9:1685-96) and the enhancers of U.S. Pat. No. 7,803,992 may alsobe used. The above list of transcriptional enhancers is not meant to belimiting. Any appropriate transcriptional enhancer can be used in theembodiments.

The termination region may be native with the transcriptional initiationregion, may be native with the operably linked DNA sequence of interest,may be native with the plant host or may be derived from another source(i.e., foreign or heterologous to the promoter, the sequence ofinterest, the plant host or any combination thereof).

Convenient termination regions are available from the Ti-plasmid of A.tumefaciens, such as the octopine synthase and nopaline synthasetermination regions. See also, Guerineau, et al., (1991) Mol. Gen.Genet. 262:141-144; Proudfoot, (1991) Cell 64:671-674; Sanfacon, et al.,(1991) Genes Dev. 5:141-149; Mogen, et al., (1990) Plant Cell2:1261-1272; Munroe, et al., (1990) Gene 91:151-158; Ballas, et al.,(1989) Nucleic Acids Res. 17:7891-7903 and Joshi, et al., (1987) NucleicAcid Res. 15:9627-9639.

Where appropriate, a nucleic acid may be optimized for increasedexpression in the host organism. Thus, where the host organism is aplant, the synthetic nucleic acids can be synthesized usingplant-preferred codons for improved expression. See, for example,Campbell and Gowri, (1990) Plant Physiol. 92:1-11 for a discussion ofhost-preferred usage. For example, although nucleic acid sequences ofthe embodiments may be expressed in both monocotyledonous anddicotyledonous plant species, sequences can be modified to account forthe specific preferences and GC content preferences of monocotyledons ordicotyledons as these preferences have been shown to differ (Murray etal. (1989) Nucleic Acids Res. 17:477-498). Thus, the maize-preferred fora particular amino acid may be derived from known gene sequences frommaize. Maize usage for 28 genes from maize plants is listed in Table 4of Murray, et al., supra. Methods are available in the art forsynthesizing plant-preferred genes. See, for example, Murray, et al.,(1989) Nucleic Acids Res. 17:477-498, and Liu H et al. Mol Bio Rep37:677-684, 2010, herein incorporated by reference. A Zea maize usagetable can be also found at kazusa.or.jp//cgi-bin/show.cgi?species=4577,which can be accessed using the www prefix. A Glycine max usage tablecan be found atkazusa.or.jp//cgi-bin/show.cgi?species=3847&aa=1&style=N, which can beaccessed using the www prefix.

In some embodiments the recombinant nucleic acid molecule encoding anIPD101 polypeptide has maize optimized codons.

Additional sequence modifications are known to enhance gene expressionin a cellular host. These include elimination of sequences encodingspurious polyadenylation signals, exon-intron splice site signals,transposon-like repeats, and other well-characterized sequences that maybe deleterious to gene expression. The GC content of the sequence may beadjusted to levels average for a given cellular host, as calculated byreference to known genes expressed in the host cell. The term “hostcell” as used herein refers to a cell which contains a vector andsupports the replication and/or expression of the expression vector isintended. Host cells may be prokaryotic cells such as E. coli oreukaryotic cells such as yeast, insect, amphibian or mammalian cells ormonocotyledonous or dicotyledonous plant cells. An example of amonocotyledonous host cell is a maize host cell. When possible, thesequence is modified to avoid predicted hairpin secondary mRNAstructures.

The expression cassettes may additionally contain 5′ leader sequences.Such leader sequences can act to enhance translation. Translationleaders are known in the art and include: picornavirus leaders, forexample, EMCV leader (Encephalomyocarditis 5′ noncoding region)(Elroy-Stein, et al., (1989) Proc. Natl. Acad. Sci. USA 86:6126-6130);potyvirus leaders, for example, TEV leader (Tobacco Etch Virus) (Gallie,et al., (1995) Gene 165(2):233-238), MDMV leader (Maize Dwarf MosaicVirus), human immunoglobulin heavy-chain binding protein (BiP) (Macejak,et al., (1991) Nature 353:90-94); untranslated leader from the coatprotein mRNA of alfalfa mosaic virus (AMV RNA 4) (Jobling, et al.,(1987) Nature 325:622-625); tobacco mosaic virus leader (TMV) (Gallie,et al., (1989) in Molecular Biology of RNA, ed. Cech (Liss, New York),pp. 237-256) and maize chlorotic mottle virus leader (MCMV) (Lommel, etal., (1991) Virology 81:382-385). See also, Della-Cioppa, et al., (1987)Plant Physiol. 84:965-968. Such constructs may also contain a “signalsequence” or “leader sequence” to facilitate co-translational orpost-translational transport of the peptide to certain intracellularstructures such as the chloroplast (or other plastid), endoplasmicreticulum or Golgi apparatus.

“Signal sequence” as used herein refers to a sequence that is known orsuspected to result in cotranslational or post-translational peptidetransport across the cell membrane. In eukaryotes, this typicallyinvolves secretion into the Golgi apparatus, with some resultingglycosylation. Insecticidal toxins of bacteria are often synthesized asprotoxins, which are proteolytically activated in the gut of the targetpest (Chang, (1987) Methods Enzymol. 153:507-516). In some embodiments,the signal sequence is located in the native sequence or may be derivedfrom a sequence of the embodiments. “Leader sequence” as used hereinrefers to any sequence that when translated, results in an amino acidsequence sufficient to trigger co-translational transport of the peptidechain to a subcellular organelle. Thus, this includes leader sequencestargeting transport and/or glycosylation by passage into the endoplasmicreticulum, passage to vacuoles, plastids including chloroplasts,mitochondria, and the like. Nuclear-encoded proteins targeted to thechloroplast thylakoid lumen compartment have a characteristic bipartitetransit peptide, composed of a stromal targeting signal peptide and alumen targeting signal peptide. The stromal targeting information is inthe amino-proximal portion of the transit peptide. The lumen targetingsignal peptide is in the carboxyl-proximal portion of the transitpeptide, and contains all the information for targeting to the lumen.Recent research in proteomics of the higher plant chloroplast hasachieved in the identification of numerous nuclear-encoded lumenproteins (Kieselbach et al. FEBS LETT 480:271-276, 2000; Peltier et al.Plant Cell 12:319-341, 2000; Bricker et al. Biochim. Biophys Acta1503:350-356, 2001), the lumen targeting signal peptide of which canpotentially be used in accordance with the present disclosure. About 80proteins from Arabidopsis, as well as homologous proteins from spinachand garden pea, are reported by Kieselbach et al., PhotosynthesisResearch, 78:249-264, 2003. In particular, Table 2 of this publication,which is incorporated into the description herewith by reference,discloses 85 proteins from the chloroplast lumen, identified by theiraccession number (see also US Patent Application Publication2009/09044298).

Suitable chloroplast transit peptides (CTP) are well known to oneskilled in the art also include chimeric CT's comprising but not limitedto, an N-terminal domain, a central domain or a C-terminal domain from aCTP from Oryza sativa 1-decoy-D xylose-5-Phosphate Synthase Oryzasativa-Superoxide dismutase Oryza sativa-soluble starch synthase Oryzasativa-NADP-dependent Malic acid enzyme Oryzasativa-Phospho-2-dehydro-3-deoxyheptonate Aldolase 2 Oryzasativa-L-Ascorbate peroxidase 5 Oryza sativa-Phosphoglucan waterdikinase, Zea Mays ssRUBISCO, Zea Mays-beta-glucosidase, Zea Mays-Malatedehydrogenase, Zea Mays Thioredoxin M-type (See US Patent ApplicationPublication 2012/0304336).

The IPD101 polypeptide gene to be targeted to the chloroplast may beoptimized for expression in the chloroplast to account for differencesin usage between the plant nucleus and this organelle. In this manner,the nucleic acids of interest may be synthesized usingchloroplast-preferred sequences.

In preparing the expression cassette, the various DNA fragments may bemanipulated so as to provide for the DNA sequences in the properorientation and, as appropriate, in the proper reading frame. Towardthis end, adapters or linkers may be employed to join the DNA fragmentsor other manipulations may be involved to provide for convenientrestriction sites, removal of superfluous DNA, removal of restrictionsites or the like. For this purpose, in vitro mutagenesis, primerrepair, restriction, annealing, resubstitutions, e.g., transitions andtransversions, may be involved.

A number of promoters can be used in the practice of the embodiments.The promoters can be selected based on the desired outcome. The nucleicacids can be combined with constitutive, tissue-preferred, inducible orother promoters for expression in the host organism. Suitableconstitutive promoters for use in a plant host cell include, forexample, the core promoter of the Rsyn7 promoter and other constitutivepromoters disclosed in WO 1999/43838 and U.S. Pat. No. 6,072,050; thecore CaMV 35S promoter (Odell, et al., (1985) Nature 313:810-812); riceactin (McElroy, et al., (1990) Plant Cell 2: 163-171); ubiquitin(Christensen, et al., (1989) Plant Mol. Biol. 12:619-632 andChristensen, et al., (1992) Plant Mol. Biol. 18:675-689); pEMU (Last, etal., (1991) Theor. Appl. Genet. 81:581-588); MAS (Velten, et al., (1984)EMBO J. 3:2723-2730); ALS promoter (U.S. Pat. No. 5,659,026) and thelike. Other constitutive promoters include, for example, those discussedin U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785;5,399,680; 5,268,463; 5,608,142 and 6,177,611.

Depending on the desired outcome, it may be beneficial to express thegene from an inducible promoter. Of particular interest for regulatingthe expression of the nucleotide sequences of the embodiments in plantsare wound-inducible promoters. Such wound-inducible promoters, mayrespond to damage caused by insect feeding, and include potatoproteinase inhibitor (pin II) gene (Ryan, (1990) Ann. Rev. Phytopath.28:425-449; Duan, et al., (1996) Nature Biotechnology 14:494-498); wun1and wun2, U.S. Pat. No. 5,428,148; win1 and win2 (Stanford, et al.,(1989) Mol. Gen. Genet. 215:200-208); systemin (McGurl, et al., (1992)Science 225:1570-1573); WIP1 (Rohmeier, et al., (1993) Plant Mol. Biol.22:783-792; Eckelkamp, et al., (1993) FEBS Letters 323:73-76); MPI gene(Corderok, et al., (1994) Plant J. 6(2):141-150) and the like.

Additionally, pathogen-inducible promoters may be employed in themethods and nucleotide constructs of the embodiments. Suchpathogen-inducible promoters include those from pathogenesis-relatedproteins (PR proteins), which are induced following infection by apathogen; e.g., PR proteins, SAR proteins, beta-1,3-glucanase,chitinase, etc. See, for example, Redolfi, et al., (1983) Neth. J. PlantPathol. 89:245-254; Uknes, et al., (1992) Plant Cell 4: 645-656 and VanLoon, (1985) Plant Mol. Virol. 4:111-116. See also, WO 1999/43819.

Of interest are promoters that are expressed locally at or near the siteof pathogen infection. See, for example, Marineau, et al., (1987) PlantMol. Biol. 9:335-342; Matton, et al., (1989) Molecular Plant-MicrobeInteractions 2:325-331; Somsisch, et al., (1986) Proc. Natl. Acad. Sci.USA 83:2427-2430; Somsisch, et al., (1988) Mol. Gen. Genet. 2:93-98 andYang, (1996) Proc. Natl. Acad. Sci. USA 93:14972-14977. See also, Chen,et al., (1996) Plant J. 10:955-966; Zhang, et al., (1994) Proc. Natl.Acad. Sci. USA 91:2507-2511; Warner, et al., (1993) Plant J. 3:191-201;Siebertz, et al., (1989) Plant Cell 1:961-968; U.S. Pat. No. 5,750,386(nematode-inducible) and the references cited therein. Of particularinterest is the inducible promoter for the maize PRms gene, whoseexpression is induced by the pathogen Fusarium moniliforme (see, forexample, Cordero, et al., (1992) Physiol. Mol. Plant Path. 41:189-200).

Chemical-regulated promoters can be used to modulate the expression of agene in a plant through the application of an exogenous chemicalregulator. Depending upon the objective, the promoter may be achemical-inducible promoter, where application of the chemical inducesgene expression or a chemical-repressible promoter, where application ofthe chemical represses gene expression. Chemical-inducible promoters areknown in the art and include, but are not limited to, the maize In2-2promoter, which is activated by benzenesulfonamide herbicide safeners,the maize GST promoter, which is activated by hydrophobic electrophiliccompounds that are used as pre-emergent herbicides, and the tobaccoPR-1a promoter, which is activated by salicylic acid. Otherchemical-regulated promoters of interest include steroid-responsivepromoters (see, for example, the glucocorticoid-inducible promoter inSchena, et al., (1991) Proc. Natl. Acad. Sci. USA 88:10421-10425 andMcNellis, et al., (1998) Plant J. 14(2):247-257) andtetracycline-inducible and tetracycline-repressible promoters (see, forexample, Gatz, et al., (1991) Mol. Gen. Genet. 227:229-237 and U.S. Pat.Nos. 5,814,618 and 5,789,156).

Tissue-preferred promoters can be utilized to target enhanced IPD101polypeptide expression within a particular plant tissue.Tissue-preferred promoters include those discussed in Yamamoto, et al.,(1997) Plant J. 12(2)255-265; Kawamata, et al., (1997) Plant CellPhysiol. 38(7):792-803; Hansen, et al., (1997) Mol. Gen Genet.254(3):337-343; Russell, et al., (1997) Transgenic Res. 6(2): 157-168;Rinehart, et al., (1996) Plant Physiol. 112(3):1331-1341; Van Camp, etal., (1996) Plant Physiol. 112(2):525-535; Canevascini, et al., (1996)Plant Physiol. 112(2):513-524; Yamamoto, et al., (1994) Plant CellPhysiol. 35(5):773-778; Lam, (1994) Results Probl. Cell Differ.20:181-196; Orozco, et al., (1993) Plant Mol Biol. 23(6):1129-1138;Matsuoka, et al., (1993) Proc Natl. Acad. Sci. USA 90(20):9586-9590 andGuevara-Garcia, et al., (1993) Plant J. 4(3):495-505. Such promoters canbe modified, if necessary, for weak expression.

Leaf-preferred promoters are known in the art. See, for example,Yamamoto, et al., (1997) Plant J. 12(2):255-265; Kwon, et al., (1994)Plant Physiol. 105:357-67; Yamamoto, et al., (1994) Plant Cell Physiol.35(5):773-778; Gotor, et al., (1993) Plant J. 3:509-18; Orozco, et al.,(1993) Plant Mol. Biol. 23(6):1129-1138 and Matsuoka, et al., (1993)Proc. Natl. Acad. Sci. USA 90(20):9586-9590.

Root-preferred or root-specific promoters are known and can be selectedfrom the many available from the literature or isolated de novo fromvarious compatible species. See, for example, Hire, et al., (1992) PlantMol. Biol. 20(2):207-218 (soybean root-specific glutamine synthetasegene); Keller and Baumgartner, (1991) Plant Cell 3(10):1051-1061(root-specific control element in the GRP 1.8 gene of French bean);Sanger, et al., (1990) Plant Mol. Biol. 14(3):433-443 (root-specificpromoter of the mannopine synthase (MAS) gene of Agrobacteriumtumefaciens) and Miao, et al., (1991) Plant Cell 3(1):11-22 (full-lengthcDNA clone encoding cytosolic glutamine synthetase (GS), which isexpressed in roots and root nodules of soybean). See also, Bogusz, etal., (1990) Plant Cell 2(7):633-641, where two root-specific promotersisolated from hemoglobin genes from the nitrogen-fixing nonlegumeParasponia andersonii and the related non-nitrogen-fixing nonlegumeTrema tomentosa are described. The promoters of these genes were linkedto a β-glucuronidase reporter gene and introduced into both thenonlegume Nicotiana tabacum and the legume Lotus corniculatus, and inboth instances root-specific promoter activity was preserved. Leach andAoyagi, (1991) describe their analysis of the promoters of the highlyexpressed rolC and rolD root-inducing genes of Agrobacterium rhizogenes(see, Plant Science (Limerick) 79(1):69-76). They concluded thatenhancer and tissue-preferred DNA determinants are dissociated in thosepromoters. Teeri, et al., (1989) used gene fusion to lacZ to show thatthe Agrobacterium T-DNA gene encoding octopine synthase is especiallyactive in the epidermis of the root tip and that the TR2′ gene is rootspecific in the intact plant and stimulated by wounding in leaf tissue,an especially desirable combination of characteristics for use with aninsecticidal or larvicidal gene (see, EMBO J. 8(2):343-350). The TR1′gene fused to nptII (neomycin phosphotransferase II) showed similarcharacteristics. Additional root-preferred promoters include theVfENOD-GRP3 gene promoter (Kuster, et al., (1995) Plant Mol. Biol.29(4):759-772) and rolB promoter (Capana, et al., (1994) Plant Mol.Biol. 25(4):681-691. See also, U.S. Pat. Nos. 5,837,876; 5,750,386;5,633,363; 5,459,252; 5,401,836; 5,110,732 and 5,023,179. Arabidopsisthaliana root-preferred regulatory sequences are disclosed inUS20130117883.

“Seed-preferred” promoters include both “seed-specific” promoters (thosepromoters active during seed development such as promoters of seedstorage proteins) as well as “seed-germinating” promoters (thosepromoters active during seed germination). See, Thompson, et al., (1989)BioEssays 10:108. Such seed-preferred promoters include, but are notlimited to, Cim1 (cytokinin-induced message); cZ19B1 (maize 19 kDazein); and mi1ps (myo-inositol-1-phosphate synthase) (see, U.S. Pat. No.6,225,529). Gamma-zein and Glb-1 are endosperm-specific promoters. Fordicots, seed-specific promoters include, but are not limited to, Kunitztrypsin inhibitor 3 (KTi3) (Jofuku and Goldberg, (1989) Plant Cell1:1079-1101), bean β-phaseolin, napin, β-conglycinin, glycinin 1,soybean lectin, cruciferin, and the like. For monocots, seed-specificpromoters include, but are not limited to, maize 15 kDa zein, 22 kDazein, 27 kDa zein, g-zein, waxy, shrunken 1, shrunken 2, globulin 1,etc. See also, WO 2000/12733, where seed-preferred promoters from end1and end2 genes are disclosed. In dicots, seed specific promoters includebut are not limited to seed coat promoter from Arabidopsis, pBAN; andthe early seed promoters from Arabidopsis, p26, p63, and p63tr (U.S.Pat. Nos. 7,294,760 and 7,847,153). A promoter that has “preferred”expression in a particular tissue is expressed in that tissue to agreater degree than in at least one other plant tissue. Sometissue-preferred promoters show expression almost exclusively in theparticular tissue.

Where low level expression is desired, weak promoters will be used.Generally, the term “weak promoter” as used herein refers to a promoterthat drives expression of a coding sequence at a low level. By low levelexpression at levels of between about 1/1000 transcripts to about1/100,000 transcripts to about 1/500,000 transcripts is intended.Alternatively, it is recognized that the term “weak promoters” alsoencompasses promoters that drive expression in only a few cells and notin others to give a total low level of expression. Where a promoterdrives expression at unacceptably high levels, portions of the promotersequence can be deleted or modified to decrease expression levels.

Such weak constitutive promoters include, for example the core promoterof the Rsyn7 promoter (WO 1999/43838 and U.S. Pat. No. 6,072,050), thecore 35S CaMV promoter, and the like. Other constitutive promotersinclude, for example, those disclosed in U.S. Pat. Nos. 5,608,149;5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463;5,608,142 and 6,177,611.

The above list of promoters is not meant to be limiting. Any appropriatepromoter can be used in the embodiments.

Generally, the expression cassette will comprise a selectable markergene for the selection of transformed cells. Selectable marker genes areutilized for the selection of transformed cells or tissues. Marker genesinclude genes encoding antibiotic resistance, such as those encodingneomycin phosphotransferase II (NEO) and hygromycin phosphotransferase(HPT), as well as genes conferring resistance to herbicidal compounds,such as glufosinate ammonium, bromoxynil, imidazolinones and2,4-dichlorophenoxyacetate (2,4-D). Additional examples of suitableselectable marker genes include, but are not limited to, genes encodingresistance to chloramphenicol (Herrera Estrella, et al., (1983) EMBO J.2:987-992); methotrexate (Herrera Estrella, et al., (1983) Nature303:209-213 and Meijer, et al., (1991) Plant Mol. Biol. 16:807-820);streptomycin (Jones, et al., (1987) Mol. Gen. Genet. 210:86-91);spectinomycin (Bretagne-Sagnard, et al., (1996) Transgenic Res.5:131-137); bleomycin (Hille, et al., (1990) Plant Mol. Biol.7:171-176); sulfonamide (Guerineau, et al., (1990) Plant Mol. Biol.15:127-136); bromoxynil (Stalker, et al., (1988) Science 242:419-423);glyphosate (Shaw, et al., (1986) Science 233:478-481 and U.S. patentapplication Ser. Nos. 10/004,357 and 10/427,692); phosphinothricin(DeBlock, et al., (1987) EMBO J. 6:2513-2518). See generally, Yarranton,(1992) Curr. Opin. Biotech. 3:506-511; Christopherson, et al., (1992)Proc. Natl. Acad. Sci. USA 89:6314-6318; Yao, et al., (1992) Cell71:63-72; Reznikoff, (1992) Mol. Microbiol. 6:2419-2422; Barkley, etal., (1980) in The Operon, pp. 177-220; Hu, et al., (1987) Cell48:555-566; Brown, et al., (1987) Cell 49:603-612; Figge, et al., (1988)Cell 52:713-722; Deuschle, et al., (1989) Proc. Natl. Acad. Sci. USA86:5400-5404; Fuerst, et al., (1989) Proc. Natl. Acad. Sci. USA86:2549-2553; Deuschle, et al., (1990) Science 248:480-483; Gossen,(1993) Ph.D. Thesis, University of Heidelberg; Reines, et al., (1993)Proc. Natl. Acad. Sci. USA 90:1917-1921; Labow, et al., (1990) Mol.Cell. Biol. 10:3343-3356; Zambretti, et al., (1992) Proc. Natl. Acad.Sci. USA 89:3952-3956; Baim, et al., (1991) Proc. Natl. Acad. Sci. USA88:5072-5076; Wyborski, et al., (1991) Nucleic Acids Res. 19:4647-4653;Hillenand-Wissman, (1989) Topics Mol. Struc. Biol. 10:143-162;Degenkolb, et al., (1991) Antimicrob. Agents Chemother. 35:1591-1595;Kleinschnidt, et al., (1988) Biochemistry 27:1094-1104; Bonin, (1993)Ph.D. Thesis, University of Heidelberg; Gossen, et al., (1992) Proc.Natl. Acad. Sci. USA 89:5547-5551; Oliva, et al., (1992) Antimicrob.Agents Chemother. 36:913-919; Hlavka, et al., (1985) Handbook ofExperimental Pharmacology, Vol. 78 (Springer-Verlag, Berlin) and Gill,et al., (1988) Nature 334:721-724.

The above list of selectable marker genes is not meant to be limiting.Any selectable marker gene can be used in the embodiments.

Plant Transformation

The methods of the embodiments involve introducing a polypeptide orpolynucleotide into a plant. “Introducing” is as used herein meanspresenting to the plant the polynucleotide or polypeptide in such amanner that the sequence gains access to the interior of a cell of theplant. The methods of the embodiments do not depend on a particularmethod for introducing a polynucleotide or polypeptide into a plant,only that the polynucleotide(s) or polypeptide(s) gains access to theinterior of at least one cell of the plant. Methods for introducingpolynucleotide(s) or polypeptide(s) into plants are known in the artincluding, but not limited to, stable transformation methods, transienttransformation methods, and virus-mediated methods.

“Stable transformation” as used herein means that the nucleotideconstruct introduced into a plant integrates into the genome of theplant and is capable of being inherited by the progeny thereof.“Transient transformation” as used herein means that a polynucleotide isintroduced into the plant and does not integrate into the genome of theplant or a polypeptide is introduced into a plant. “Plant” as usedherein refers to whole plants, plant organs (e.g., leaves, stems, roots,etc.), seeds, plant cells, propagules, embryos and progeny of the same.Plant cells can be differentiated or undifferentiated (e.g. callus,suspension culture cells, protoplasts, leaf cells, root cells, phloemcells and pollen).

Transformation protocols as well as protocols for introducing nucleotidesequences into plants may vary depending on the type of plant or plantcell, i.e., monocot or dicot, targeted for transformation. Suitablemethods of introducing nucleotide sequences into plant cells andsubsequent insertion into the plant genome include microinjection(Crossway, et al., (1986) Biotechniques 4:320-334), electroporation(Riggs, et al., (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606),Agrobacterium-mediated transformation (U.S. Pat. Nos. 5,563,055 and5,981,840), direct gene transfer (Paszkowski, et al., (1984) EMBO J.3:2717-2722) and ballistic particle acceleration (see, for example, U.S.Pat. Nos. 4,945,050; 5,879,918; 5,886,244 and 5,932,782; Tomes, et al.,(1995) in Plant Cell, Tissue, and Organ Culture: Fundamental Methods,ed. Gamborg and Phillips, (Springer-Verlag, Berlin) and McCabe, et al.,(1988) Biotechnology 6:923-926) and Lec1 transformation (WO 00/28058).For potato transformation see, Tu, et al., (1998) Plant MolecularBiology 37:829-838 and Chong, et al., (2000) Transgenic Research9:71-78. Additional transformation procedures can be found inWeissinger, et al., (1988) Ann. Rev. Genet. 22:421-477; Sanford, et al.,(1987) Particulate Science and Technology 5:27-37 (onion); Christou, etal., (1988) Plant Physiol. 87:671-674 (soybean); McCabe, et al., (1988)Bio/Technology 6:923-926 (soybean); Finer and McMullen, (1991) In VitroCell Dev. Biol. 27P:175-182 (soybean); Singh, et al., (1998) Theor.Appl. Genet. 96:319-324 (soybean); Datta, et al., (1990) Biotechnology8:736-740 (rice); Klein, et al., (1988) Proc. Natl. Acad. Sci. USA85:4305-4309 (maize); Klein, et al., (1988) Biotechnology 6:559-563(maize); U.S. Pat. Nos. 5,240,855; 5,322,783 and 5,324,646; Klein, etal., (1988) Plant Physiol. 91:440-444 (maize); Fromm, et al., (1990)Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren, et al., (1984)Nature (London) 311:763-764; U.S. Pat. No. 5,736,369 (cereals);Bytebier, et al., (1987) Proc. Natl. Acad. Sci. USA 84:5345-5349(Liliaceae); De Wet, et al., (1985) in The Experimental Manipulation ofOvule Tissues, ed. Chapman, et al., (Longman, N.Y.), pp. 197-209(pollen); Kaeppler, et al., (1990) Plant Cell Reports 9:415-418 andKaeppler, et al., (1992) Theor. Appl. Genet. 84:560-566(whisker-mediated transformation); D'Halluin, et al., (1992) Plant Cell4:1495-1505 (electroporation); Li, et al., (1993) Plant Cell Reports12:250-255 and Christou and Ford, (1995) Annals of Botany 75:407-413(rice); Osjoda, et al., (1996) Nature Biotechnology 14:745-750 (maizevia Agrobacterium tumefaciens).

In specific embodiments, the sequences of the embodiments can beprovided to a plant using a variety of transient transformation methods.Such transient transformation methods include, but are not limited to,the introduction of the IPD101 polynucleotide or variants and fragmentsthereof directly into the plant or the introduction of the IPD101polypeptide transcript into the plant. Such methods include, forexample, microinjection or particle bombardment. See, for example,Crossway, et al., (1986) Mol Gen. Genet. 202:179-185; Nomura, et al.,(1986) Plant Sci. 44:53-58; Hepler, et al., (1994) Proc. Natl. Acad.Sci. 91:2176-2180 and Hush, et al., (1994) The Journal of Cell Science107:775-784. Alternatively, the IPD101 polynucleotide can be transientlytransformed into the plant using techniques known in the art. Suchtechniques include viral vector system and the precipitation of thepolynucleotide in a manner that precludes subsequent release of the DNA.Thus, transcription from the particle-bound DNA can occur, but thefrequency with which it is released to become integrated into the genomeis greatly reduced. Such methods include the use of particles coatedwith polyethylimine (PEI; Sigma #P3143).

Methods are known in the art for the targeted insertion of apolynucleotide at a specific location in the plant genome. In oneembodiment, the insertion of the polynucleotide at a desired genomiclocation is achieved using a site-specific recombination system. See,for example, WO 1999/25821, WO 1999/25854, WO 1999/25840, WO 1999/25855and WO 1999/25853. Briefly, the polynucleotide of the embodiments can becontained in transfer cassette flanked by two non-identicalrecombination sites. The transfer cassette is introduced into a planthave stably incorporated into its genome a target site which is flankedby two non-identical recombination sites that correspond to the sites ofthe transfer cassette. An appropriate recombinase is provided and thetransfer cassette is integrated at the target site. The polynucleotideof interest is thereby integrated at a specific chromosomal position inthe plant genome.

Plant transformation vectors may be comprised of one or more DNA vectorsneeded for achieving plant transformation. For example, it is a commonpractice in the art to utilize plant transformation vectors that arecomprised of more than one contiguous DNA segment. These vectors areoften referred to in the art as “binary vectors”. Binary vectors as wellas vectors with helper plasmids are most often used forAgrobacterium-mediated transformation, where the size and complexity ofDNA segments needed to achieve efficient transformation is quite large,and it is advantageous to separate functions onto separate DNAmolecules. Binary vectors typically contain a plasmid vector thatcontains the cis-acting sequences required for T-DNA transfer (such asleft border and right border), a selectable marker that is engineered tobe capable of expression in a plant cell, and a “gene of interest” (agene engineered to be capable of expression in a plant cell for whichgeneration of transgenic plants is desired). Also present on thisplasmid vector are sequences required for bacterial replication. Thecis-acting sequences are arranged in a fashion to allow efficienttransfer into plant cells and expression therein. For example, theselectable marker gene and the pesticidal gene are located between theleft and right borders. Often a second plasmid vector contains thetrans-acting factors that mediate T-DNA transfer from Agrobacterium toplant cells. This plasmid often contains the virulence functions (Virgenes) that allow infection of plant cells by Agrobacterium, andtransfer of DNA by cleavage at border sequences and vir-mediated DNAtransfer, as is understood in the art (Hellens and Mullineaux, (2000)Trends in Plant Science 5:446-451). Several types of Agrobacteriumstrains (e.g. LBA4404, GV3101, EHA101, EHA105, etc.) can be used forplant transformation. The second plasmid vector is not necessary fortransforming the plants by other methods such as microprojection,microinjection, electroporation, polyethylene glycol, etc.

In general, plant transformation methods involve transferringheterologous DNA into target plant cells (e.g., immature or matureembryos, suspension cultures, undifferentiated callus, protoplasts,etc.), followed by applying a maximum threshold level of appropriateselection (depending on the selectable marker gene) to recover thetransformed plant cells from a group of untransformed cell mass.Following integration of heterologous foreign DNA into plant cells, onethen applies a maximum threshold level of appropriate selection in themedium to kill the untransformed cells and separate and proliferate theputatively transformed cells that survive from this selection treatmentby transferring regularly to a fresh medium. By continuous passage andchallenge with appropriate selection, one identifies and proliferatesthe cells that are transformed with the plasmid vector. Molecular andbiochemical methods can then be used to confirm the presence of theintegrated heterologous gene of interest into the genome of thetransgenic plant.

Explants are typically transferred to a fresh supply of the same mediumand cultured routinely. Subsequently, the transformed cells aredifferentiated into shoots after placing on regeneration mediumsupplemented with a maximum threshold level of selecting agent. Theshoots are then transferred to a selective rooting medium for recoveringrooted shoot or plantlet. The transgenic plantlet then grows into amature plant and produces fertile seeds (e.g., Hiei, et al., (1994) ThePlant Journal 6:271-282; Ishida, et al., (1996) Nature Biotechnology14:745-750). Explants are typically transferred to a fresh supply of thesame medium and cultured routinely. A general description of thetechniques and methods for generating transgenic plants are found inAyres and Park, (1994) Critical Reviews in Plant Science 13:219-239 andBommineni and Jauhar, (1997) Maydica 42:107-120. Since the transformedmaterial contains many cells; both transformed and non-transformed cellsare present in any piece of subjected target callus or tissue or groupof cells. The ability to kill non-transformed cells and allowtransformed cells to proliferate results in transformed plant cultures.Often, the ability to remove non-transformed cells is a limitation torapid recovery of transformed plant cells and successful generation oftransgenic plants.

The cells that have been transformed may be grown into plants inaccordance with conventional ways. See, for example, McCormick, et al.,(1986) Plant Cell Reports 5:81-84. These plants may then be grown, andeither pollinated with the same transformed strain or different strains,and the resulting hybrid having constitutive or inducible expression ofthe desired phenotypic characteristic identified. Two or moregenerations may be grown to ensure that expression of the desiredphenotypic characteristic is stably maintained and inherited and thenseeds harvested to ensure that expression of the desired phenotypiccharacteristic has been achieved.

The nucleotide sequences of the embodiments may be provided to the plantby contacting the plant with a virus or viral nucleic acids. Generally,such methods involve incorporating the nucleotide construct of interestwithin a viral DNA or RNA molecule. It is recognized that therecombinant proteins of the embodiments may be initially synthesized aspart of a viral polyprotein, which later may be processed by proteolysisin vivo or in vitro to produce the desired IPD101 polypeptide. It isalso recognized that such a viral polyprotein, comprising at least aportion of the amino acid sequence of an IPD101 polypeptide of theembodiments, may have the desired pesticidal activity. Such viralpolyproteins and the nucleotide sequences that encode for them areencompassed by the embodiments. Methods for providing plants withnucleotide constructs and producing the encoded proteins in the plants,which involve viral DNA or RNA molecules, are known in the art. See, forexample, U.S. Pat. Nos. 5,889,191; 5,889,190; 5,866,785; 5,589,367 and5,316,931.

Methods for transformation of chloroplasts are known in the art. See,for example, Svab, et al., (1990) Proc. Natl. Acad. Sci. USA87:8526-8530; Svab and Maliga, (1993) Proc. Natl. Acad. Sci. USA90:913-917; Svab and Maliga, (1993) EMBO J. 12:601-606. The methodrelies on particle gun delivery of DNA containing a selectable markerand targeting of the DNA to the plastid genome through homologousrecombination. Additionally, plastid transformation can be accomplishedby transactivation of a silent plastid-borne transgene bytissue-preferred expression of a nuclear-encoded and plastid-directedRNA polymerase. Such a system has been reported in McBride, et al.,(1994) Proc. Natl. Acad. Sci. USA 91:7301-7305.

The embodiments further relate to plant-propagating material of atransformed plant of the embodiments including, but not limited to,seeds, tubers, corms, bulbs, leaves and cuttings of roots and shoots.

The embodiments may be used for transformation of any plant species,including, but not limited to, monocots and dicots. Examples of plantsof interest include, but are not limited to, corn (Zea mays), Brassicasp. (e.g., B. napus, B. rapa, B. juncea), particularly those Brassicaspecies useful as sources of seed oil, alfalfa (Medicago sativa), rice(Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghumvulgare), millet (e.g., pearl millet (Pennisetum glaucum), proso millet(Panicum miliaceum), foxtail millet (Setaria italica), finger millet(Eleusine coracana)), sunflower (Helianthus annuus), safflower(Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycinemax), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts(Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum),sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee(Coffea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus),citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camelliasinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficuscasica), guava (Psidium guajava), mango (Mangifera indica), olive (Oleaeuropaea), papaya (Carica papaya), cashew (Anacardium occidentale),macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugarbeets (Beta vulgaris), sugarcane (Saccharum spp.), oats, barley,vegetables ornamentals, and conifers.

Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g.,Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseoluslimensis), peas (Lathyrus spp.), and members of the genus Cucumis suchas cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon(C. melo). Ornamentals include azalea (Rhododendron spp.), hydrangea(Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosaspp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias(Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia(Euphorbia pulcherrima), and chrysanthemum. Conifers that may beemployed in practicing the embodiments include, for example, pines suchas loblolly pine (Pinus taeda), slash pine (Pinus elliotii), ponderosapine (Pinus ponderosa), lodgepole pine (Pinus contorta), and Montereypine (Pinus radiata); Douglas-fir (Pseudotsuga menziesii); Westernhemlock (Tsuga canadensis); Sitka spruce (Picea glauca); redwood(Sequoia sempervirens); true firs such as silver fir (Abies amabilis)and balsam fir (Abies balsamea); and cedars such as Western red cedar(Thuja plicata) and Alaska yellow-cedar (Chamaecyparis nootkatensis).Plants of the embodiments include crop plants (for example, corn,alfalfa, sunflower, Brassica, soybean, cotton, safflower, peanut,sorghum, wheat, millet, tobacco, etc.), such as corn and soybean plants.

Turf grasses include, but are not limited to: annual bluegrass (Poaannua); annual ryegrass (Lolium multiflorum); Canada bluegrass (Poacompressa); Chewing's fescue (Festuca rubra); colonial bentgrass(Agrostis tenuis); creeping bentgrass (Agrostis palustris); crestedwheatgrass (Agropyron desertorum); fairway wheatgrass (Agropyroncristatum); hard fescue (Festuca longifolia); Kentucky bluegrass (Poapratensis); orchardgrass (Dactylis glomerata); perennial ryegrass(Lolium perenne); red fescue (Festuca rubra); redtop (Agrostis alba);rough bluegrass (Poa trivialis); sheep fescue (Festuca ovina); smoothbromegrass (Bromus inermis); tall fescue (Festuca arundinacea); timothy(Phleum pratense); velvet bentgrass (Agrostis carina); weepingalkaligrass (Puccinellia distans); western wheatgrass (Agropyronsmithii); Bermuda grass (Cynodon spp.); St. Augustine grass(Stenotaphrum secundatum); zoysia grass (Zoysia spp.); Bahia grass(Paspalum notatum); carpet grass (Axonopus affinis); centipede grass(Eremochloa ophiuroides); kikuyu grass (Pennisetum clandesinum);seashore paspalum (Paspalum vaginatum); blue gramma (Boutelouagracilis); buffalo grass (Buchloe dactyloids); sideoats gramma(Bouteloua curtipendula).

Plants of interest include grain plants that provide seeds of interest,oil-seed plants, and leguminous plants. Seeds of interest include grainseeds, such as corn, wheat, barley, rice, sorghum, rye, millet, etc.Oil-seed plants include cotton, soybean, safflower, sunflower, Brassica,maize, alfalfa, palm, coconut, flax, castor, olive, etc. Leguminousplants include beans and peas. Beans include guar, locust bean,fenugreek, soybean, garden beans, cowpea, mung bean, lima bean, favabean, lentils, chickpea, etc.

Following introduction of heterologous foreign DNA into plant cells, thetransformation or integration of heterologous gene in the plant genomeis confirmed by various methods such as analysis of nucleic acids,proteins and metabolites associated with the integrated gene.

PCR analysis is a rapid method to screen transformed cells, tissue orshoots for the presence of incorporated gene at the earlier stage beforetransplanting into the soil (Sambrook and Russell, (2001) MolecularCloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.). PCR is carried out using oligonucleotide primersspecific to the gene of interest or Agrobacterium vector background,etc.

Plant transformation may be confirmed by Southern blot analysis ofgenomic DNA (Sambrook and Russell, (2001) supra). In Northern blotanalysis, RNA is isolated from specific tissues of transformant,fractionated in a formaldehyde agarose gel, and blotted onto a nylonfilter according to standard procedures that are routinely used in theart (Sambrook and Russell, (2001) supra). Expression of RNA encoded bythe pesticidal gene is then tested by hybridizing the filter to aradioactive probe derived from a pesticidal gene, by methods known inthe art (Sambrook and Russell, (2001) supra). Western blot, biochemicalassays and the like may be carried out on the transgenic plants toconfirm the presence of protein encoded by the pesticidal gene bystandard procedures (Sambrook and Russell, 2001, supra) using antibodiesthat bind to one or more epitopes present on the IPD101 polypeptide.

Methods to Introduce Genome Editing Technologies into Plants

In some embodiments, the disclosed IPD101 polynucleotide compositionscan be introduced into the genome of a plant using genome editingtechnologies, or previously introduced IPD101 polynucleotides in thegenome of a plant may be edited using genome editing technologies. Forexample, the disclosed polynucleotides can be introducted into a desiredlocation in the genome of a plant through the use of double-strandedbreak technologies such as TALENs, meganucleases, zinc finger nucleases,CRISPR-Cas, and the like. For example, the disclosed polynucleotides canbe introduced into a desired location in a genome using a CRISPR-Cassystem, for the purpose of site-specific insertion. The desired locationin a plant genome can be any desired target site for insertion, such asa genomic region amenable for breeding or may be a target site locatedin a genomic window with an existing trait of interest. Existing traitsof interest could be either an endogenous trait or a previouslyintroduced trait.

In some embodiments, where the disclosed IPD101 polynucleotide haspreviously been introduced into a genome, genome editing technologiesmay be used to alter or modify the introduced polynucleotide sequence.Site specific modifications that can be introduced into the disclosedIPD101 polynucleotide compositions include those produced using anymethod for introducing site specific modification, including, but notlimited to, through the use of gene repair oligonucleotides (e.g. USPublication 2013/0019349), or through the use of double-stranded breaktechnologies such as TALENs, meganucleases, zinc finger nucleases,CRISPR-Cas, and the like. Such technologies can be used to modify thepreviously introduced polynucleotide through the insertion, deletion orsubstitution of nucleotides within the introduced polynucleotide.Alternatively, double-stranded break technologies can be used to addadditional nucleotide sequences to the introduced polynucleotide.Additional sequences that may be added include, additional expressionelements, such as enhancer and promoter sequences. In anotherembodiment, genome editing technologies may be used to positionadditional insecticidally-active proteins in close proximity to thedisclosed IPD101 polynucleotide compositions disclosed herein within thegenome of a plant, in order to generate molecular stacks ofinsecticidally-active proteins.

An “altered target site,” “altered target sequence.” “modified targetsite,” and “modified target sequence” are used interchangeably hereinand refer to a target sequence as disclosed herein that comprises atleast one alteration when compared to non-altered target sequence. Such“alterations” include, for example: (i) replacement of at least onenucleotide, (ii) a deletion of at least one nucleotide, (iii) aninsertion of at least one nucleotide, or (iv) any combination of(i)-(iii).

Stacking of Traits in Transgenic Plant

Transgenic plants may comprise a stack of one or more insecticidalpolynucleotides disclosed herein with one or more additionalpolynucleotides resulting in the production or suppression of multiplepolypeptide sequences. Transgenic plants comprising stacks ofpolynucleotide sequences can be obtained by either or both oftraditional breeding methods or through genetic engineering methods.These methods include, but are not limited to, breeding individual lineseach comprising a polynucleotide of interest, transforming a transgenicplant comprising a gene disclosed herein with a subsequent gene andco-transformation of genes into a single plant cell. As used herein, theterm “stacked” includes having the multiple traits present in the sameplant (i.e., both traits are incorporated into the nuclear genome, onetrait is incorporated into the nuclear genome and one trait isincorporated into the genome of a plastid or both traits areincorporated into the genome of a plastid). In one non-limiting example,“stacked traits” comprise a molecular stack where the sequences arephysically adjacent to each other. A trait, as used herein, refers tothe phenotype derived from a particular sequence or groups of sequences.Co-transformation of genes can be carried out using singletransformation vectors comprising multiple genes or genes carriedseparately on multiple vectors. If the sequences are stacked bygenetically transforming the plants, the polynucleotide sequences ofinterest can be combined at any time and in any order. The traits can beintroduced simultaneously in a co-transformation protocol with thepolynucleotides of interest provided by any combination oftransformation cassettes. For example, if two sequences will beintroduced, the two sequences can be contained in separatetransformation cassettes (trans) or contained on the same transformationcassette (cis). Expression of the sequences can be driven by the samepromoter or by different promoters. In certain cases, it may bedesirable to introduce a transformation cassette that will suppress theexpression of the polynucleotide of interest. This may be combined withany combination of other suppression cassettes or overexpressioncassettes to generate the desired combination of traits in the plant. Itis further recognized that polynucleotide sequences can be stacked at adesired genomic location using a site-specific recombination system.See, for example, WO 1999/25821, WO 1999/25854, WO 1999/25840, WO1999/25855 and WO 1999/25853, all of which are herein incorporated byreference.

In some embodiments, one or more of the polynucleotides encoding theIPD101 polypeptide(s) disclosed herein, alone or stacked with one ormore additional insect resistance traits can be stacked with one or moreadditional input traits (e.g., herbicide resistance, fungal resistance,virus resistance, stress tolerance, disease resistance, male sterility,stalk strength, and the like) or output traits (e.g., increased yield,modified starches, improved oil profile, balanced amino acids, highlysine or methionine, increased digestibility, improved fiber quality,drought resistance, and the like). Thus, the polynucleotide embodimentscan be used to provide a complete agronomic package of improved cropquality with the ability to flexibly and cost effectively control anynumber of agronomic pests.

Transgenes useful for stacking include but are not limited to:transgenes that confer resistance to a herbicide; transgenes that conferor contribute to an altered grain characteristic; genes that controlmale-sterility; genes that create a site for site specific dnaintegration; genes that affect abiotic stress resistance; genes thatconfer increased yield genes that confer plant digestibility; andtransgenes that confer resistance to insects or disease.

Examples of transgenes that confer resistance to insects include genesencoding a Bacillus thuringiensis protein, a derivative thereof or asynthetic polypeptide modeled thereon. See, for example, Geiser, et al.,(1986) Gene 48:109, who disclose the cloning and nucleotide sequence ofa Bt delta-endotoxin gene. Moreover, DNA molecules encodingdelta-endotoxin genes can be purchased from American Type CultureCollection (Rockville, Md.), for example, under ATCC® Accession Numbers40098, 67136, 31995 and 31998. Other non-limiting examples of Bacillusthuringiensis transgenes being genetically engineered are given in thefollowing patents and patent applications: U.S. Pat. Nos. 5,188,960;5,689,052; 5,880,275; 5,986,177; 6,023,013, 6,060,594, 6,063,597,6,077,824, 6,620,988, 6,642,030, 6,713,259, 6,893,826, 7,105,332;7,179,965, 7,208,474; 7,227,056, 7,288,643, 7,323,556, 7,329,736,7,449,552, 7,468,278, 7,510,878, 7,521,235, 7,544,862, 7,605,304,7,696,412, 7,629,504, 7,705,216, 7,772,465, 7,790,846, 7,858,849 and WO1991/14778; WO 1999/31248; WO 2001/12731; WO 1999/24581 and WO1997/40162.

Genes encoding pesticidal proteins may also be stacked including but arenot limited to: insecticidal proteins from Pseudomonas sp. such asPSEEN3174 (Monalysin, (2011) PLoS Pathogens, 7:1-13), from Pseudomonasprotegees strain CHAO and Pf-5 (previously fluorescens) (Pechy-Tarr,(2008) Environmental Microbiology 10:2368-2386: GenBank Accession No.EU400157); from Pseudomonas taiwanensis (Liu, et al., (2010) J. Agric.Food Chem. 58:12343-12349) and from Pseudomonas pseudoalcaligenes(Zhang, et al., (2009) Annals of Microbiology 59:45-50 and Li, et al.,(2007) Plant Cell Tiss. Organ Cult. 89:159-168); insecticidal proteinsfrom Photorhabdus sp. and Xenorhabdus sp. (Hinchliffe, et al., (2010)The Open Toxinology Journal 3:101-118 and Morgan, et al., (2001) Appliedand Envir. Micro. 67:2062-2069), U.S. Pat. Nos. 6,048,838, and6,379,946; a PIP-1 polypeptide of US Patent Application PublicationNumber US20140007292; an AfIP-1A and/or AfIP-1B polypeptide of US PatentApplication Publication Number US20140033361; a PHI-4 polypeptide of USPatent Application Publication Number US20140274885 and US20160040184; aPIP-47 polypeptide of US Patent Application Publication NumberUS20160186204, a PIP-72 polypeptide of US Patent Application PublicationNumber US20160366891; a PtIP-50 polypeptide and a PtIP-65 polypeptide ofUS Patent Application Publication Number 20170166921; a PtIP-83polypeptide of US Patent Application Publication Number 20160347799; aPtIP-96 polypeptide of US Patent Application Publication Number20170233440; an IPD079 polypeptide of U.S. Ser. No. 62/201,977; anIPD082 polypeptide of U.S. Ser. No. 62/269,482, and 6-endotoxinsincluding, but not limited to, the Cry1, Cry2, Cry3, Cry4, Cry5, Cry6,Cry7, Cry8, Cry9, Cry10, Cry11, Cry12, Cry13, Cry14, Cry15, Cry16,Cry17, Cry18, Cry19, Cry20, Cry21, Cry22, Cry23, Cry24, Cry25, Cry26,Cry27, Cry 28, Cry 29, Cry 30, Cry31, Cry32, Cry33, Cry34, Cry35, Cry36,Cry37, Cry38, Cry39, Cry40, Cry41, Cry42, Cry43, Cry44, Cry45, Cry 46,Cry47, Cry49, Cry50, Cry51, Cry52, Cry53, Cry 54, Cry55, Cry56, Cry57,Cry58, Cry59, Cry60, Cry61, Cry62, Cry63, Cry64, Cry65, Cry66, Cry67,Cry68, Cry69, Cry70, Cry71, and Cry 72 classes of δ-endotoxin genes andthe B. thuringiensis cytolytic Cyt1 and Cyt2 genes. Members of theseclasses of B. thuringiensis insecticidal proteins well known to oneskilled in the art (see, Crickmore, et al., “Bacillus thuringiensistoxin nomenclature” (2011), atlifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/ which can be accessed onthe world-wide web using the “www” prefix).

Examples of δ-endotoxins also include but are not limited to Cry1Aproteins of U.S. Pat. Nos. 5,880,275 and 7,858,849; a DIG-3 or DIG-11toxin (N-terminal deletion of α-helix 1 and/or α-helix 2 variants of Cryproteins such as Cry1A) of U.S. Pat. Nos. 8,304,604 and 8,304,605, Cry1Bof U.S. patent application Ser. No. 10/525,318; Cry1C of U.S. Pat. No.6,033,874; Cry1F of U.S. Pat. Nos. 5,188,960, 6,218,188; Cry1A/Fchimeras of U.S. Pat. Nos. 7,070,982; 6,962,705 and 6,713,063); a Cry2protein such as Cry2Ab protein of U.S. Pat. No. 7,064,249); a Cry3Aprotein including but not limited to an engineered hybrid insecticidalprotein (eHIP) created by fusing unique combinations of variable regionsand conserved blocks of at least two different Cry proteins (US PatentApplication Publication Number 2010/0017914); a Cry4 protein; a Cry5protein; a Cry6 protein; Cry8 proteins of U.S. Pat. Nos. 7,329,736,7,449,552, 7,803,943, 7,476,781, 7,105,332, 7,378,499 and 7,462,760; aCry9 protein such as such as members of the Cry9A, Cry9B, Cry9C, Cry9D,Cry9E, and Cry9F families; a Cry15 protein of Naimov, et al., (2008)Applied and Environmental Microbiology 74:7145-7151; a Cry22, a Cry34Ab1protein of U.S. Pat. Nos. 6,127,180, 6,624,145 and 6,340,593; a CryET33and CryET34 protein of U.S. Pat. Nos. 6,248,535, 6,326,351, 6,399,330,6,949,626, 7,385,107 and 7,504,229; a CryET33 and CryET34 homologs of USPatent Publication Number 2006/0191034, 2012/0278954, and PCTPublication Number WO 2012/139004; a Cry35Ab1 protein of U.S. Pat. Nos.6,083,499, 6,548,291 and 6,340,593; a Cry46 protein, a Cry 51 protein, aCry binary toxin; a TIC901 or related toxin; TIC807 of US 2008/0295207;ET29, ET37, TIC809, TIC810, TIC812, TIC127, TIC128 of PCT US2006/033867; AXMI-027, AXMI-036, and AXMI-038 of U.S. Pat. No.8,236,757; AXMI-031, AXMI-039, AXMI-040, AXMI-049 of U.S. Pat. No.7,923,602; AXMI-018, AXMI-020, and AXMI-021 of WO 2006/083891; AXMI-010of WO 2005/038032; AXMI-003 of WO 2005/021585; AXMI-008 of US2004/0250311; AXMI-006 of US 2004/0216186; AXMI-007 of US 2004/0210965;AXMI-009 of US 2004/0210964; AXMI-014 of US 2004/0197917; AXMI-004 of US2004/0197916; AXMI-028 and AXMI-029 of WO 2006/119457; AXMI-007,AXMI-008, AXMI-0080rf2, AXMI-009, AXMI-014 and AXMI-004 of WO2004/074462; AXMI-150 of U.S. Pat. No. 8,084,416; AXMI-205 ofUS20110023184; AXMI-011, AXMI-012, AXMI-013, AXMI-015, AXMI-019,AXMI-044, AXMI-037, AXMI-043, AXMI-033, AXMI-034, AXMI-022, AXMI-023,AXMI-041, AXMI-063, and AXMI-064 of US 2011/0263488; AXMI-R1 and relatedproteins of US 2010/0197592; AXMI221Z, AXMI222z, AXMI223z, AXMI224z andAXMI225z of WO 2011/103248; AXMI218, AXMI219, AXMI220, AXMI226, AXMI227,AXMI228, AXMI229, AXMI230, and AXMI231 of WO11/103247; AXMI-115,AXMI-113, AXMI-005, AXMI-163 and AXMI-184 of U.S. Pat. No. 8,334,431;AXMI-001, AXMI-002, AXMI-030, AXMI-035, and AXMI-045 of US 2010/0298211;AXMI-066 and AXMI-076 of US2009/0144852; AXMI128, AXMI130, AXMI131,AXMI133, AXMI140, AXMI141, AXMI142, AXMI143, AXMI144, AXMI146, AXMI148,AXMI149, AXMI152, AXMI153, AXMI154, AXMI155, AXMI156, AXMI157, AXMI158,AXMI162, AXMI165, AXMI166, AXMI167, AXMI168, AXMI169, AXMI170, AXMI171,AXMI172, AXMI173, AXMI174, AXMI175, AXMI176, AXMI177, AXMI178, AXMI179,AXMI180, AXMI181, AXMI182, AXMI185, AXMI186, AXMI187, AXMI188, AXMI189of U.S. Pat. No. 8,318,900; AXMI079, AXMI080, AXMI081, AXMI082, AXMI091,AXMI092, AXMI096, AXMI097, AXMI098, AXMI099, AXMI100, AXMI101, AXMI102,AXMI103, AXMI104, AXMI107, AXMI108, AXMI109, AXMI110, AXMI111, AXMI112,AXMI114, AXMI116, AXMI117, AXMI118, AXMI119, AXMI120, AXMI121, AXMI122,AXMI123, AXMI124, AXMI1257, AXMI1268, AXMI127, AXMI129, AXMI164,AXMI151, AXMI161, AXMI183, AXMI132, AXMI138, AXMI137 of US 2010/0005543;and Cry proteins such as Cry1A and Cry3A having modified proteolyticsites of U.S. Pat. No. 8,319,019; and a Cry1Ac, Cry2Aa and Cry1Ca toxinprotein from Bacillus thuringiensis strain VBTS 2528 of US PatentApplication Publication Number 2011/0064710. Other Cry proteins are wellknown to one skilled in the art (see, Crickmore, et al., “Bacillusthuringiensis toxin nomenclature” (2011), atlifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/ which can be accessed onthe world-wide web using the “www” prefix). The insecticidal activity ofCry proteins is well known to one skilled in the art (for review, see,van Frannkenhuyzen, (2009) J. Invert. Path. 101:1-16). The use of Cryproteins as transgenic plant traits is well known to one skilled in theart and Cry-transgenic plants including but not limited to Cry1Ac,Cry1Ac+Cry2Ab, Cry1Ab, Cry1A.105, Cry1F, Cry1Fa2, Cry1F+Cry1Ac, Cry2Ab,Cry3A, mCry3A, Cry3Bb1, Cry34Ab1, Cry35Ab1, Vip3A, mCry3A, Cry9c andCBI-Bt have received regulatory approval (see, Sanahuja, (2011) PlantBiotech Journal 9:283-300 and the CERA (2010) GM Crop Database Centerfor Environmental Risk Assessment (CERA), ILSI Research Foundation,Washington D.C. at cera-gmc.org/index.php?action=gm_crop_database whichcan be accessed on the world-wide web using the “www” prefix). More thanone pesticidal proteins well known to one skilled in the art can also beexpressed in plants such as Vip3Ab & Cry1Fa (US2012/0317682), Cry1BE &Cry1F (US2012/0311746), Cry1CA & Cry1AB (US2012/0311745), Cry1F & CryCa(US2012/0317681), Cry1DA & Cry1BE (US2012/0331590), Cry1DA & Cry1Fa(US2012/0331589), Cry1AB & Cry1BE (US2012/0324606), and Cry1Fa & Cry2Aa,Cry1I or Cry1E (US2012/0324605). Pesticidal proteins also includeinsecticidal lipases including lipid acyl hydrolases of U.S. Pat. No.7,491,869, and cholesterol oxidases such as from Streptomyces (Purcellet al. (1993) Biochem Biophys Res Commun 15:1406-1413). Pesticidalproteins also include VIP (vegetative insecticidal proteins) toxins ofU.S. Pat. Nos. 5,877,012, 6,107,279, 6,137,033, 7,244,820, 7,615,686,and 8,237,020, and the like. Other VIP proteins are well known to oneskilled in the art (see,lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/vip.html which can beaccessed on the world-wide web using the “www” prefix). Pesticidalproteins also include toxin complex (TC) proteins, obtainable fromorganisms such as Xenorhabdus, Photorhabdus and Paenibacillus (see, U.S.Pat. Nos. 7,491,698 and 8,084,418). Some TC proteins have “stand alone”insecticidal activity and other TC proteins enhance the activity of thestand-alone toxins produced by the same given organism. The toxicity ofa “stand-alone” TC protein (from Photorhabdus, Xenorhabdus orPaenibacillus, for example) can be enhanced by one or more TC protein“potentiators” derived from a source organism of a different genus.There are three main types of TC proteins. As referred to herein, ClassA proteins (“Protein A”) are stand-alone toxins. Class B proteins(“Protein B”) and Class C proteins (“Protein C”) enhance the toxicity ofClass A proteins. Examples of Class A proteins are TcbA, TcdA, XptA1 andXptA2. Examples of Class B proteins are TcaC, TcdB, XptB1Xb and XptC1Wi.Examples of Class C proteins are TccC, XptC1Xb and XptB1Wi. Pesticidalproteins also include spider, snake and scorpion venom proteins.Examples of spider venom peptides include but are not limited tolycotoxin-1 peptides and mutants thereof (U.S. Pat. No. 8,334,366).

Further transgenes that confer resistance to insects may down-regulationof expression of target genes in insect pest species by interferingribonucleic acid (RNA) molecules through RNA interference. RNAinterference refers to the process of sequence-specificpost-transcriptional gene silencing in animals mediated by shortinterfering RNAs (siRNAs) (Fire, et al., (1998) Nature 391:806). RNAitransgenes may include but are not limited to expression of dsRNA,siRNA, miRNA, iRNA, antisense RNA, or sense RNA molecules thatdown-regulate expression of target genes in insect pests. PCTPublication WO 2007/074405 describes methods of inhibiting expression oftarget genes in invertebrate pests including Colorado potato beetle. PCTPublication WO 2005/110068 describes methods of inhibiting expression oftarget genes in invertebrate pests including in particular Western cornrootworm as a means to control insect infestation. Furthermore, PCTPublication WO 2009/091864 describes compositions and methods for thesuppression of target genes from insect pest species including pestsfrom the Lygus genus.

RNAi transgenes are provieded for targeting the vacuolar ATPase Hsubunit, useful for controlling a coleopteran pest population andinfestation as described in US Patent Application Publication2012/0198586. PCT Publication WO 2012/055982 describes ribonucleic acid(RNA or double stranded RNA) that inhibits or down regulates theexpression of a target gene that encodes: an insect ribosomal proteinsuch as the ribosomal protein L19, the ribosomal protein L40 or theribosomal protein S27A; an insect proteasome subunit such as the Rpn6protein, the Pros 25, the Rpn2 protein, the proteasome beta 1 subunitprotein or the Pros beta 2 protein; an insect β-coatomer of the COPIvesicle, the γ-coatomer of the COPI vesicle, the β′-coatomer protein orthe ζ-coatomer of the COPI vesicle; an insect Tetraspanine 2 A proteinwhich is a putative transmembrane domain protein; an insect proteinbelonging to the actin family such as Actin 5C; an insect ubiquitin-5Eprotein; an insect Sec23 protein which is a GTPase activator involved inintracellular protein transport; an insect crinkled protein which is anunconventional myosin which is involved in motor activity; an insectcrooked neck protein which is involved in the regulation of nuclearalternative mRNA splicing; an insect vacuolar H+-ATPase G-subunitprotein and an insect Tbp-1 such as Tat-binding protein. PCT publicationWO 2007/035650 describes ribonucleic acid (RNA or double stranded RNA)that inhibits or down regulates the expression of a target gene thatencodes Snf7. US Patent Application publication 2011/0054007 describespolynucleotide silencing elements targeting RPS10. US Patent Applicationpublication 2014/0275208 and US2015/0257389 describes polynucleotidesilencing elements targeting RyanR and PAT3. PCT publicationsWO/2016/138106, WO 2016/060911, WO 2016/060912, WO 2016/060913, and WO2016/060914 describe polynucleotide silencing elements targeting COPIcoatomer subunit nucleic acid molecules that confer resistance toColeopteran and Hemipteran pests. US Patent Application Publications2012/029750, US 20120297501, and 2012/0322660 describe interferingribonucleic acids (RNA or double stranded RNA) that functions uponuptake by an insect pest species to down-regulate expression of a targetgene in said insect pest, wherein the RNA comprises at least onesilencing element wherein the silencing element is a region ofdouble-stranded RNA comprising annealed complementary strands, onestrand of which comprises or consists of a sequence of nucleotides whichis at least partially complementary to a target nucleotide sequencewithin the target gene. US Patent Application Publication 2012/0164205describe potential targets for interfering double stranded ribonucleicacids for inhibiting invertebrate pests including: a Chd3 HomologousSequence, a Beta-Tubulin Homologous Sequence, a 40 kDa V-ATPaseHomologous Sequence, a EF1α Homologous Sequence, a 26S ProteosomeSubunit p28 Homologous Sequence, a Juvenile Hormone Epoxide HydrolaseHomologous Sequence, a Swelling Dependent Chloride Channel ProteinHomologous Sequence, a Glucose-6-Phosphate 1-Dehydrogenase ProteinHomologous Sequence, an Act42A Protein Homologous Sequence, aADP-Ribosylation Factor 1 Homologous Sequence, a Transcription FactorIIB Protein Homologous Sequence, a Chitinase Homologous Sequences, aUbiquitin Conjugating Enzyme Homologous Sequence, aGlyceraldehyde-3-Phosphate Dehydrogenase Homologous Sequence, anUbiquitin B Homologous Sequence, a Juvenile Hormone Esterase Homolog,and an Alpha Tubuliln Homologous Sequence.

Use in Pesticidal Control

General methods for employing strains comprising a nucleic acid sequenceof the embodiments or a variant thereof, in pesticide control or inengineering other organisms as pesticidal agents are known in the art.

Microorganism hosts that are known to occupy the “phytosphere”(phylloplane, phyllosphere, rhizosphere, and/or rhizoplana) of one ormore crops of interest may be selected. These microorganisms areselected so as to be capable of successfully competing in the particularenvironment with the wild-type microorganisms, provide for stablemaintenance and expression of the gene(s) expressing one or more of theIPD101 polypeptides and desirably provide for improved protection of thepesticide from environmental degradation and inactivation.

Alternatively, the IPD101 polypeptide is produced by introducing aheterologous gene into a cellular host. Expression of the heterologousgene results, directly or indirectly, in the intracellular productionand maintenance of the pesticide. These cells are then treated underconditions that prolong the activity of the toxin produced in the cellwhen the cell is applied to the environment of target pest(s). Theresulting product retains the toxicity of the toxin. These naturallyencapsulated IPD101 polypeptides may then be formulated in accordancewith conventional techniques for application to the environment hostinga target pest, e.g., soil, water, and foliage of plants. See, forexample EPA 0192319, and the references cited therein.

Pesticidal Compositions

In some embodiments the active ingredients can be applied in the form ofcompositions and can be applied to the crop area or plant to be treated,simultaneously or in succession, with other compounds. These compoundscan be fertilizers, weed killers, Cryoprotectants, surfactants,detergents, pesticidal soaps, dormant oils, polymers, and/ortime-release or biodegradable carrier formulations that permit long-termdosing of a target area following a single application of theformulation. They can also be selective herbicides, chemicalinsecticides, virucides, microbicides, amoebicides, pesticides,fungicides, bacteriocides, nematocides, molluscicides or mixtures ofseveral of these preparations, if desired, together with furtheragriculturally acceptable carriers, surfactants or application-promotingadjuvants customarily employed in the art of formulation. Suitablecarriers and adjuvants can be solid or liquid and correspond to thesubstances ordinarily employed in formulation technology, e.g. naturalor regenerated mineral substances, solvents, dispersants, wettingagents, tackifiers, binders or fertilizers. Likewise, the formulationsmay be prepared into edible “baits” or fashioned into pest “traps” topermit feeding or ingestion by a target pest of the pesticidalformulation.

Methods of applying an active ingredient or an agrochemical compositionthat contains at least one of the IPD101 polypeptide(s) produced by thebacterial strains include leaf application, seed coating and soilapplication. The number of applications and the rate of applicationdepend on the intensity of infestation by the corresponding pest.

The composition may be formulated as a powder, dust, pellet, granule,spray, emulsion, colloid, solution or such like, and may be prepared bysuch conventional means as desiccation, lyophilization, homogenation,extraction, filtration, centrifugation, sedimentation or concentrationof a culture of cells comprising the polypeptide. In all suchcompositions that contain at least one such pesticidal polypeptide, thepolypeptide may be present in a concentration of from about 1% to about99% by weight.

Lepidopteran, Dipteran, Heteropteran, nematode, Hemiptera or Coleopteranpests may be killed or reduced in numbers in a given area by the methodsof the disclosure or may be prophylactically applied to an environmentalarea to prevent infestation by a susceptible pest. Preferably the pestingests or is contacted with, a pesticidally-effective amount of thepolypeptide. “Pesticidally-effective amount” as used herein refers to anamount of the pesticide that is able to bring about death to at leastone pest or to noticeably reduce pest growth, feeding or normalphysiological development. This amount will vary depending on suchfactors as, for example, the specific target pests to be controlled, thespecific environment, location, plant, crop or agricultural site to betreated, the environmental conditions and the method, rate,concentration, stability, and quantity of application of thepesticidally-effective polypeptide composition. The formulations mayalso vary with respect to climatic conditions, environmentalconsiderations, and/or frequency of application and/or severity of pestinfestation.

The pesticide compositions described may be made by formulating eitherthe bacterial cell, Crystal and/or spore suspension or isolated proteincomponent with the desired agriculturally-acceptable carrier. Thecompositions may be formulated prior to administration in an appropriatemeans such as lyophilized, freeze-dried, desiccated or in an aqueouscarrier, medium or suitable diluent, such as saline or other buffer. Theformulated compositions may be in the form of a dust or granularmaterial or a suspension in oil (vegetable or mineral) or water oroil/water emulsions or as a wettable powder or in combination with anyother carrier material suitable for agricultural application. Suitableagricultural carriers can be solid or liquid and are well known in theart. The term “agriculturally-acceptable carrier” covers all adjuvants,inert components, dispersants, surfactants, tackifiers, binders, etc.that are ordinarily used in pesticide formulation technology; these arewell known to those skilled in pesticide formulation. The formulationsmay be mixed with one or more solid or liquid adjuvants and prepared byvarious means, e.g., by homogeneously mixing, blending and/or grindingthe pesticidal composition with suitable adjuvants using conventionalformulation techniques. Suitable formulations and application methodsare described in U.S. Pat. No. 6,468,523. The plants can also be treatedwith one or more chemical compositions, including one or more herbicide,insecticides or fungicides. Exemplary chemical compositions include:Fruits/Vegetables Herbicides: Atrazine, Bromacil, Diuron, Glyphosate,Linuron, Metribuzin, Simazine, Trifluralin, Fluazifop, Glufosinate, Halosulfuron Gowan, Paraquat, Propyzamide, Sethoxydim, Butafenacil,Halosulfuron, Indaziflam; Fruits/Vegetables Insecticides: Aldicarb,Bacillus thuriengiensis, Carbaryl, Carbofuran, Chlorpyrifos,Cypermethrin, Deltamethrin, Diazinon, Malathion, Abamectin,Cyfluthrin/beta-cyfluthrin, Esfenvalerate, Lambda-cyhalothrin,Acequinocyl, Bifenazate, Methoxyfenozide, Novaluron, Chromafenozide,Thiacloprid, Dinotefuran, FluaCrypyrim, Tolfenpyrad, Clothianidin,Spirodiclofen, Gamma-cyhalothrin, Spiromesifen, Spinosad, Rynaxypyr,Cyazypyr, Spinoteram, Triflumuron, Spirotetramat, Imidacloprid,Flubendiamide, Thiodicarb, Metaflumizone, Sulfoxaflor, Cyflumetofen,Cyanopyrafen, Imidacloprid, Clothianidin, Thiamethoxam, Spinotoram,Thiodicarb, Flonicamid, Methiocarb, Emamectin-benzoate, Indoxacarb,Forthiazate, Fenamiphos, Cadusaphos, Pyriproxifen, Fenbutatin-oxid,Hexthiazox, Methomyl,4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on;Fruits/Vegetables Fungicides: Carbendazim, Chlorothalonil, EBDCs,Sulphur, Thiophanate-methyl, Azoxystrobin, Cymoxanil, Fluazinam,Fosetyl, Iprodione, Kresoxim-methyl, Metalaxyl/mefenoxam,Trifloxystrobin, Ethaboxam, Iprovalicarb, Trifloxystrobin, Fenhexamid,Oxpoconazole fumarate, Cyazofamid, Fenamidone, Zoxamide, Picoxystrobin,Pyraclostrobin, Cyflufenamid, Boscalid; Cereals Herbicides: Isoproturon,Bromoxynil, Ioxynil, Phenoxies, Chlorsulfuron, Clodinafop, Diclofop,Diflufenican, Fenoxaprop, Florasulam, Fluoroxypyr, Metsulfuron,Triasulfuron, Flucarbazone, Iodosulfuron, Propoxycarbazone, Picolinafen,Mesosulfuron, Beflubutamid, Pinoxaden, Amidosulfuron, ThifensulfuronMethyl, Tribenuron, Flupyrsulfuron, Sulfosulfuron, Pyrasulfotole,Pyroxsulam, Flufenacet, Tralkoxydim, Pyroxasulfon; Cereals Fungicides:Carbendazim, Chlorothalonil, Azoxystrobin, Cyproconazole, Cyprodinil,Fenpropimorph, Epoxiconazole, Kresoxim-methyl, Quinoxyfen, Tebuconazole,Trifloxystrobin, Simeconazole, Picoxystrobin, Pyraclostrobin,Dimoxystrobin, Prothioconazole, Fluoxastrobin; Cereals Insecticides:Dimethoate, Lambda-cyhalthrin, Deltamethrin, alpha-Cypermethrin,β-cyfluthrin, Bifenthrin, Imidacloprid, Clothianidin, Thiamethoxam,Thiacloprid, Acetamiprid, Dinetofuran, Clorphyriphos, Metamidophos,Oxidemethon-methyl, Pirimicarb, Methiocarb; Maize Herbicides: Atrazine,Alachlor, Bromoxynil, Acetochlor, Dicamba, Clopyralid, (S-)Dimethenamid, Glufosinate, Glyphosate, Isoxaflutole, (S-)Metolachlor,Mesotrione, Nicosulfuron, Primisulfuron, Rimsulfuron, Sulcotrione,Foramsulfuron, Topramezone, Tembotrione, Saflufenacil, Thiencarbazone,Flufenacet, Pyroxasulfon; Maize Insecticides: Carbofuran, Chlorpyrifos,Bifenthrin, Fipronil, Imidacloprid, Lambda-Cyhalothrin, Tefluthrin,Terbufos, Thiamethoxam, Clothianidin, Spiromesifen, Flubendiamide,Triflumuron, Rynaxypyr, Deltamethrin, Thiodicarb, β-Cyfluthrin,Cypermethrin, Bifenthrin, Lufenuron, Triflumoron, Tefluthrin,Tebupirimphos, Ethiprole, Cyazypyr, Thiacloprid, Acetamiprid,Dinetofuran, Avermectin, Methiocarb, Spirodiclofen, Spirotetramat; MaizeFungicides: Fenitropan, Thiram, Prothioconazole, Tebuconazole,Trifloxystrobin; Rice Herbicides: Butachlor, Propanil, Azimsulfuron,Bensulfuron, Cyhalofop, Daimuron, Fentrazamide, Imazosulfuron,Mefenacet, Oxaziclomefone, Pyrazosulfuron, Pyributicarb, Quinclorac,Thiobencarb, Indanofan, Flufenacet, Fentrazamide, Halosulfuron,Oxaziclomefone, Benzobicyclon, Pyriftalid, Penoxsulam, Bispyribac,Oxadiargyl, Ethoxysulfuron, Pretilachlor, Mesotrione, Tefuryltrione,Oxadiazone, Fenoxaprop, Pyrimisulfan; Rice Insecticides: Diazinon,Fenitrothion, Fenobucarb, Monocrotophos, Benfuracarb, Buprofezin,Dinotefuran, Fipronil, Imidacloprid, Isoprocarb, Thiacloprid,Chromafenozide, Thiacloprid, Dinotefuran, Clothianidin, Ethiprole,Flubendiamide, Rynaxypyr, Deltamethrin, Acetamiprid, Thiamethoxam,Cyazypyr, Spinosad, Spinotoram, Emamectin-Benzoate, Cypermethrin,Chlorpyriphos, Cartap, Methamidophos, Etofenprox, Triazophos,4-[[((6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on,Carbofuran, Benfuracarb; Rice Fungicides: Thiophanate-methyl,Azoxystrobin, Carpropamid, Edifenphos, Ferimzone, Iprobenfos,Isoprothiolane, Pencycuron, Probenazole, Pyroquilon, Tricyclazole,Trifloxystrobin, Diclocymet, Fenoxanil, Simeconazole, Tiadinil; CottonHerbicides: Diuron, Fluometuron, MSMA, Oxyfluorfen, Prometryn,Trifluralin, Carfentrazone, Clethodim, Fluazifop-butyl, Glyphosate,Norflurazon, Pendimethalin, Pyrithiobac-sodium, Trifloxysulfuron,Tepraloxydim, Glufosinate, Flumioxazin, Thidiazuron; CottonInsecticides: Acephate, Aldicarb, Chlorpyrifos, Cypermethrin,Deltamethrin, Malathion, Monocrotophos, Abamectin, Acetamiprid,Emamectin Benzoate, Imidacloprid, Indoxacarb, Lambda-Cyhalothrin,Spinosad, Thiodicarb, Gamma-Cyhalothrin, Spiromesifen, Pyridalyl,Flonicamid, Flubendiamide, Triflumuron, Rynaxypyr, Beta-Cyfluthrin,Spirotetramat, Clothianidin, Thiamethoxam, Thiacloprid, Dinetofuran,Flubendiamide, Cyazypyr, Spinosad, Spinotoram, gamma Cyhalothrin,4-[[(((6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on,Thiodicarb, Avermectin, Flonicamid, Pyridalyl, Spiromesifen,Sulfoxaflor, Profenophos, Thriazophos, Endosulfan; Cotton Fungicides:Etridiazole, Metalaxyl, Quintozene; Soybean Herbicides: Alachlor,Bentazone, Trifluralin, Chlorimuron-Ethyl, Cloransulam-Methyl,Fenoxaprop, Fomesafen, Fluazifop, Glyphosate, Imazamox, Imazaquin,Imazethapyr, (S-)Metolachlor, Metribuzin, Pendimethalin, Tepraloxydim,Glufosinate; Soybean Insecticides: Lambda-cyhalothrin, Methomyl,Parathion, Thiocarb, Imidacloprid, Clothianidin, Thiamethoxam,Thiacloprid, Acetamiprid, Dinetofuran, Flubendiamide, Rynaxypyr,Cyazypyr, Spinosad, Spinotoram, Emamectin-Benzoate, Fipronil, Ethiprole,Deltamethrin, β-Cyfluthrin, gamma and lambda Cyhalothrin,4-[[(((6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on,Spirotetramat, Spinodiclofen, Triflumuron, Flonicamid, Thiodicarb,beta-Cyfluthrin; Soybean Fungicides: Azoxystrobin, Cyproconazole,Epoxiconazole, Flutriafol, Pyraclostrobin, Tebuconazole,Trifloxystrobin, Prothioconazole, Tetraconazole; Sugarbeet Herbicides:Chloridazon, Desmedipham, Ethofumesate, Phenmedipham, Triallate,Clopyralid, Fluazifop, Lenacil, Metamitron, Quinmerac, Cycloxydim,Triflusulfuron, Tepraloxydim, Quizalofop; Sugarbeet Insecticides:Imidacloprid, Clothianidin, Thiamethoxam, Thiacloprid, Acetamiprid,Dinetofuran, Deltamethrin, β-Cyfluthrin, gamma/lambda Cyhalothrin,4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on,Tefluthrin, Rynaxypyr, Cyaxypyr, Fipronil, Carbofuran; CanolaHerbicides: Clopyralid, Diclofop, Fluazifop, Glufosinate, Glyphosate,Metazachlor, Trifluralin Ethametsulfuron, Quinmerac, Quizalofop,Clethodim, Tepraloxydim; Canola Fungicides: Azoxystrobin, Carbendazim,Fludioxonil, Iprodione, Prochloraz, Vinclozolin; Canola Insecticides:Carbofuran organophosphates, Pyrethroids, Thiacloprid, Deltamethrin,Imidacloprid, Clothianidin, Thiamethoxam, Acetamiprid, Dinetofuran,β-Cyfluthrin, gamma and lambda Cyhalothrin, tau-Fluvaleriate, Ethiprole,Spinosad, Spinotoram, Flubendiamide, Rynaxypyr, Cyazypyr,4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on.

In some embodiments the herbicide is Atrazine, Bromacil, Diuron,Chlorsulfuron, Metsulfuron, Thifensulfuron Methyl, Tribenuron,Acetochlor, Dicamba, Isoxaflutole, Nicosulfuron, Rimsulfuron,Pyrithiobac-sodium, Flumioxazin, Chlorimuron-Ethyl, Metribuzin,Quizalofop, S-metolachlor, Hexazinne or combinations thereof.

In some embodiments the insecticide is Esfenvalerate,Chlorantraniliprole, Methomyl, Indoxacarb, Oxamyl or combinationsthereof.

Pesticidal and Insecticidal Activity

“Pest” includes but is not limited to, insects, fungi, bacteria,nematodes, mites, ticks and the like. Insect pests include insectsselected from the orders Coleoptera, Diptera, Hymenoptera, Lepidoptera,Mallophaga, Homoptera, Hemiptera Orthroptera, Thysanoptera, Dermaptera,Isoptera, Anoplura, Siphonaptera, Trichoptera, etc., particularlyLepidoptera and Coleoptera.

Those skilled in the art will recognize that not all compounds areequally effective against all pests. Compounds of the embodimentsdisplay activity against insect pests, which may include economicallyimportant agronomic, forest, greenhouse, nursery ornamentals, food andfiber, public and animal health, domestic and commercial structure,household and stored product pests.

Larvae of the order Lepidoptera include, but are not limited to,armyworms, cutworms, loopers and heliothines in the family NoctuidaeSpodoptera frugiperda JE Smith (fall armyworm); S. exigua Hubner (beetarmyworm); S. litura Fabricius (tobacco cutworm, cluster caterpillar);Mamestra configurata Walker (bertha armyworm); M. brassicae Linnaeus(cabbage moth); Agrotis ipsilon Hufnagel (black cutworm); A. orthogoniaMorrison (western cutworm); A. subterranea Fabricius (granulatecutworm); Alabama argillacea Hubner (cotton leaf worm); Trichoplusia niHubner (cabbage looper); Pseudoplusia includens Walker (soybean looper);Anticarsia gemmatalis Hubner (velvetbean caterpillar); Hypena scabraFabricius (green cloverworm); Heliothis virescens Fabricius (tobaccobudworm); Pseudaletia unipuncta Haworth (armyworm); Athetis mindaraBarnes and Mcdunnough (rough skinned cutworm); Euxoa messoria Harris(darksided cutworm); Earias insulana Boisduval (spiny bollworm); E.vittella Fabricius (spotted bollworm); Helicoverpa armigera Hubner(American bollworm); H. zea Boddie (corn earworm or cotton bollworm);Melanchra picta Harris (zebra caterpillar); Egira (Xylomyges) curialisGrote (citrus cutworm); borers, casebearers, webworms, coneworms, andskeletonizers from the family Pyralidae Ostrinia nubilalis Hubner(European corn borer); Amyelois transitella Walker (naval orangeworm);Anagasta kuehniella Zeller (Mediterranean flour moth); Cadra cautellaWalker (almond moth); Chilo suppressalis Walker (rice stem borer); C.partellus, (sorghum borer); Corcyra cephalonica Stainton (rice moth);Crambus caliginosellus Clemens (corn root webworm); C. teterrellusZincken (bluegrass webworm); Cnaphalocrocis medinalis Guenee (rice leafroller); Desmia funeralis Hubner (grape leaffolder); Diaphania hyalinataLinnaeus (melon worm); D. nitidalis Stoll (pickleworm); Diatraeagrandiosella Dyar (southwestern corn borer), D. saccharalis Fabricius(surgarcane borer); Eoreuma loftini Dyar (Mexican rice borer); Ephestiaelutella Hubner (tobacco (cacao) moth); Galleria mellonella Linnaeus(greater wax moth); Herpetogramma licarsisalis Walker (sod webworm);Homoeosoma electellum Hulst (sunflower moth); Elasmopalpus lignosellusZeller (lesser cornstalk borer); Achroia grisella Fabricius (lesser waxmoth); Loxostege sticticalis Linnaeus (beet webworm); Orthaga thyrisalisWalker (tea tree web moth); Maruca testulalis Geyer (bean pod borer);Plodia interpunctella Hubner (Indian meal moth); Scirpophaga incertulasWalker (yellow stem borer); Udea rubigalis Guenee (celery leaftier); andleafrollers, budworms, seed worms and fruit worms in the familyTortricidae Acleris gloverana Walsingham (Western blackheaded budworm);A. variana Fernald (Eastern blackheaded budworm); Archips argyrospilaWalker (fruit tree leaf roller); A. rosana Linnaeus (European leafroller); and other Archips species, Adoxophyes orana Fischer vonRosslerstamm (summer fruit tortrix moth); Cochylis hospes Walsingham(banded sunflower moth); Cydia latiferreana Walsingham (filbertworm); C.pomonella Linnaeus (coding moth); Platynota flavedana Clemens(variegated leafroller); P. stultana Walsingham (omnivorous leafroller);Lobesia botrana Denis & Schiffermiffier (European grape vine moth);Spilonota ocellana Denis & Schiffermiffier (eyespotted bud moth);Endopiza viteana Clemens (grape berry moth); Eupoecilia ambiguellaHubner (vine moth); Bonagota salubricola Meyrick (Brazilian appleleafroller); Grapholita molesta Busck (oriental fruit moth); Suleimahelianthana Riley (sunflower bud moth); Argyrotaenia spp.; Choristoneuraspp.

Selected other agronomic pests in the order Lepidoptera include, but arenot limited to, Alsophila pometaria Harris (fall cankerworm); Anarsialineatella Zeller (peach twig borer); Anisota senatoria J. E. Smith(orange striped oakworm); Antheraea pernyi Guérin-Méneville (Chinese OakTussah Moth); Bombyx mori Linnaeus (Silkworm); Bucculatrix thurberiellaBusck (cotton leaf perforator); Colias eurytheme Boisduval (alfalfacaterpillar); Datana integerrima Grote & Robinson (walnut caterpillar);Dendrolimus sibiricus Tschetwerikov (Siberian silk moth), Ennomossubsignaria Hubner (elm spanworm); Erannis tiliaria Harris (lindenlooper); Euproctis chrysorrhoea Linnaeus (browntail moth); Harrisinaamericana Guérin-Méneville (grapeleaf skeletonizer); Hemileuca oliviaeCockrell (range caterpillar); Hyphantria cunea Drury (fall webworm);Keiferia lycopersicella Walsingham (tomato pinworm); Lambdinafiscellaria fiscellaria Hulst (Eastern hemlock looper); L. fiscellarialugubrosa Hulst (Western hemlock looper); Leucoma salicis Linnaeus(satin moth); Lymantria dispar Linnaeus (gypsy moth); Manducaquinquemaculata Haworth (five spotted hawk moth, tomato hornworm); M.sexta Haworth (tomato hornworm, tobacco hornworm); Operophtera brumataLinnaeus (winter moth); Paleacrita vernata Peck (spring cankerworm);Papilio cresphontes Cramer (giant swallowtail orange dog); Phryganidiacalifornica Packard (California oakworm); Phyllocnistis citrellaStainton (citrus leafminer); Phyllonorycter blancardella Fabricius(spotted tentiform leafminer); Pieris brassicae Linnaeus (large whitebutterfly); P. rapae Linnaeus (small white butterfly); P. caapi Linnaeus(green veined white butterfly); Platyptilia carduidactyla Riley(artichoke plume moth); Plutella xylostella Linnaeus (diamondback moth);Pectinophora gossypiella Saunders (pink bollworm); Pontia protodiceBoisduval and Leconte (Southern cabbageworm); Sabulodes aegrotata Guenee(omnivorous looper); Schizura concinna J. E. Smith (red humpedcaterpillar); Sitotroga cerealella Olivier (Angoumois grain moth);Thaumetopoea pityocampa Schiffermuller (pine processionary caterpillar);Tineola bisselliella Hummel (webbing clothesmoth); Tuta absoluta Meyrick(tomato leafminer); Yponomeuta padella Linnaeus (ermine moth); Heliothissubflexa Guenee; Malacosoma spp. and Orgyia spp.

Of interest are larvae and adults of the order Coleoptera includingweevils from the families Anthribidae, Bruchidae and Curculionidae(including, but not limited to: Anthonomus grandis Boheman (bollweevil); Lissorhoptrus oryzophilus Kuschel (rice water weevil);Sitophilus granarius Linnaeus (granary weevil); S. oryzae Linnaeus (riceweevil); Hypera punctata Fabricius (clover leaf weevil);Cylindrocopturus adspersus LeConte (sunflower stem weevil); Smicronyxfulvus LeConte (red sunflower seed weevil); S. sordidus LeConte (graysunflower seed weevil); Sphenophorus maidis Chittenden (maize billbug));flea beetles, cucumber beetles, rootworms, leaf beetles, potato beetlesand leafminers in the family Chrysomelidae (including, but not limitedto: Leptinotarsa decemlineata Say (Colorado potato beetle); Diabroticavirgifera virgifera LeConte (western corn rootworm); D. barberi Smithand Lawrence (northern corn rootworm); D. undecimpunctata howardi Barber(southern corn rootworm); Chaetocnema pulicaria Melsheimer (corn fleabeetle); Phyllotreta cruciferae Goeze (Crucifer flea beetle);Phyllotreta striolata (stripped flea beetle); Colaspis brunnea Fabricius(grape colaspis); Oulema melanopus Linnaeus (cereal leaf beetle);Zygogramma exclamationis Fabricius (sunflower beetle)); beetles from thefamily Coccinellidae (including, but not limited to: Epilachnavarivestis Mulsant (Mexican bean beetle)); chafers and other beetlesfrom the family Scarabaeidae (including, but not limited to: Popilliajaponica Newman (Japanese beetle); Cyclocephala borealis Arrow (northernmasked chafer, white grub); C. immaculata Olivier (southern maskedchafer, white grub); Rhizotrogus majalis Razoumowsky (European chafer);Phyllophaga crinita Burmeister (white grub); Ligyrus gibbosus De Geer(carrot beetle)); carpet beetles from the family Dermestidae; wirewormsfrom the family Elateridae, Eleodes spp., Melanotus spp.; Conoderusspp.; Limonius spp.; Agriotes spp.; Ctenicera spp.; Aeolus spp.; barkbeetles from the family Scolytidae and beetles from the familyTenebrionidae.

Adults and immatures of the order Diptera are of interest, includingleafminers Agromyza parvicornis Loew (corn blotch leafminer); midges(including, but not limited to: Contarinia sorghicola Coquillett(sorghum midge); Mayetiola destructor Say (Hessian fly); Sitodiplosismosellana Gain (wheat midge); Neolasioptera murtfeldtiana Felt,(sunflower seed midge)); fruit flies (Tephritidae), Oscinella fritLinnaeus (fruit flies); maggots (including, but not limited to: Deliaplatura Meigen (seedcorn maggot); D. coarctata Fallen (wheat bulb fly)and other Delia spp., Meromyza americana Fitch (wheat stem maggot);Musca domestica Linnaeus (house flies); Fannia canicularis Linnaeus, F.femoralis Stein (lesser house flies); Stomoxys calcitrans Linnaeus(stable flies)); face flies, horn flies, blow flies, Chrysomya spp.;Phormia spp. and other muscoid fly pests, horse flies Tabanus spp.; botflies Gastrophilus spp.; Oestrus spp.; cattle grubs Hypoderma spp.; deerflies Chrysops spp.; Melophagus ovinus Linnaeus (keds) and otherBrachycera, mosquitoes Aedes spp.; Anopheles spp.; Culex spp.; blackflies Prosimulium spp.; Simulium spp.; biting midges, sand flies,sciarids, and other Nematocera.

Included as insects of interest are adults and nymphs of the ordersHemiptera and Homoptera such as, but not limited to, adelgids from thefamily Adelgidae, plant bugs from the family Miridae, cicadas from thefamily Cicadidae, leafhoppers, Empoasca spp.; from the familyCicadellidae, planthoppers from the families Cixiidae, Flatidae,Fulgoroidea, Issidae and Delphacidae, treehoppers from the familyMembracidae, psyllids from the family Psyllidae, whiteflies from thefamily Aleyrodidae, aphids from the family Aphididae, phylloxera fromthe family Phylloxeridae, mealybugs from the family Pseudococcidae,scales from the families Asterolecanidae, Coccidae, Dactylopiidae,Diaspididae, Eriococcidae Ortheziidae, Phoenicococcidae andMargarodidae, lace bugs from the family Tingidae, stink bugs from thefamily Pentatomidae, cinch bugs, Blissus spp.; and other seed bugs fromthe family Lygaeidae, spittlebugs from the family Cercopidae squash bugsfrom the family Coreidae and red bugs and cotton stainers from thefamily Pyrrhocoridae.

Agronomically important members from the order Homoptera furtherinclude, but are not limited to: Acyrthisiphon pisum Harris (pea aphid);Aphis craccivora Koch (cowpea aphid); A. fabae Scopoli (black beanaphid); A. gossypii Glover (cotton aphid, melon aphid); A. maidiradicisForbes (corn root aphid); A. pomi De Geer (apple aphid); A. spiraecolaPatch (spirea aphid); Aulacorthum solani Kaltenbach (foxglove aphid);Chaetosiphon fragaefolii Cockerell (strawberry aphid); Diuraphis noxiaKurdjumov/Mordvilko (Russian wheat aphid); Dysaphis plantagineaPaaserini (rosy apple aphid); Eriosoma lanigerum Hausmann (woolly appleaphid); Brevicoryne brassicae Linnaeus (cabbage aphid); Hyalopteruspruni Geoffroy (mealy plum aphid); Lipaphis erysimi Kaltenbach (turnipaphid); Metopolophium dirrhodum Walker (cereal aphid); Macrosiphumeuphorbiae Thomas (potato aphid); Myzus persicae Sulzer (peach-potatoaphid, green peach aphid); Nasonovia ribisnigri Mosley (lettuce aphid);Pemphigus spp. (root aphids and gall aphids); Rhopalosiphum maidis Fitch(corn leaf aphid); R. padi Linnaeus (bird cherry-oat aphid); Schizaphisgraminum Rondani (greenbug); Sipha flava Forbes (yellow sugarcaneaphid); Sitobion avenae Fabricius (English grain aphid); Therioaphismaculata Buckton (spotted alfalfa aphid); Toxoptera aurantii Boyer deFonscolombe (black citrus aphid) and T. citricida Kirkaldy (brown citrusaphid); Adelges spp. (adelgids); Phylloxera devastatrix Pergande (pecanphylloxera); Bemisia tabaci Gennadius (tobacco whitefly, sweetpotatowhitefly); B. argentifolii Bellows & Perring (silverleaf whitefly);Dialeurodes citri Ashmead (citrus whitefly); Trialeurodes abutiloneus(bandedwinged whitefly) and T. vaporariorum Westwood (greenhousewhitefly); Empoasca fabae Harris (potato leafhopper); Laodelphaxstriatellus Fallen (smaller brown planthopper); Macrolestesquadrilineatus Forbes (aster leafhopper); Nephotettix cinticeps Uhler(green leafhopper); N. nigropictus Stat (rice leafhopper); Nilaparvatalugens Stat (brown planthopper); Peregrinus maidis Ashmead (cornplanthopper); Sogatella furcifera Horvath (white-backed planthopper);Sogatodes orizicola Muir (rice delphacid); Typhlocyba pomaria McAtee(white apple leafhopper); Erythroneoura spp. (grape leafhoppers);Magicicada septendecim Linnaeus (periodical cicada); Icerya purchasiMaskell (cottony cushion scale); Quadraspidiotus perniciosus Comstock(San Jose scale); Planococcus citri Risso (citrus mealybug);Pseudococcus spp. (other mealybug complex); Cacopsylla pyricola Foerster(pear psylla); Trioza diospyri Ashmead (persimmon psylla).

Agronomically important species of interest from the order Hemipterainclude, but are not limited to: Acrosternum hilare Say (green stinkbug); Anasa tristis De Geer (squash bug); Blissus leucopterusleucopterus Say (chinch bug); Corythuca gossypii Fabricius (cotton lacebug); Cyrtopeltis modesta Distant (tomato bug); Dysdercus suturellusHerrich-Schäffer (cotton stainer); Euschistus servus Say (brown stinkbug); E. variolarius Palisot de Beauvois (one-spotted stink bug);Graptostethus spp. (complex of seed bugs); Leptoglossus corculus Say(leaf-footed pine seed bug); Lygus lineolaris Palisot de Beauvois(tarnished plant bug); L. Hesperus Knight (Western tarnished plant bug);L. pratensis Linnaeus (common meadow bug); L. rugulipennis Poppius(European tarnished plant bug); Lygocoris pabulinus Linnaeus (commongreen capsid); Nezara viridula Linnaeus (southern green stink bug);Oebalus pugnax Fabricius (rice stink bug); Oncopeltus fasciatus Dallas(large milkweed bug); Pseudatomoscelis seriatus Reuter (cottonfleahopper).

Furthermore, embodiments may be effective against Hemiptera such,Calocoris norvegicus Gmelin (strawberry bug); Orthops campestrisLinnaeus; Plesiocoris rugicollis Fallen (apple capsid); Cyrtopeltismodestus Distant (tomato bug); Cyrtopeltis notatus Distant (suckfly);Spanagonicus albofasciatus Reuter (whitemarked fleahopper); Diaphnocorischlorionis Say (honeylocust plant bug); Labopidicola allii Knight (onionplant bug); Pseudatomoscelis seriatus Reuter (cotton fleahopper);Adelphocoris rapidus Say (rapid plant bug); Poecilocapsus lineatusFabricius (four-lined plant bug); Nysius ericae Schilling (false chinchbug); Nysius raphanus Howard (false chinch bug); Nezara viridulaLinnaeus (Southern green stink bug); Eurygaster spp.; Coreidae spp.;Pyrrhocoridae spp.; Tinidae spp.; Blostomatidae spp.; Reduviidae spp.and Cimicidae spp.

Also included are adults and larvae of the order Acari (mites) such asAceria tosichella Keifer (wheat curl mite); Petrobia latens Müller(brown wheat mite); spider mites and red mites in the familyTetranychidae, Panonychus ulmi Koch (European red mite); Tetranychusurticae Koch (two spotted spider mite); (T. mcdanieli McGregor (McDanielmite); T. cinnabarinus Boisduval (carmine spider mite); T. turkestaniUgarov & Nikolski (strawberry spider mite); flat mites in the familyTenuipalpidae, Brevipalpus lewisi McGregor (citrus flat mite); rust andbud mites in the family Eriophyidae and other foliar feeding mites andmites important in human and animal health, i.e., dust mites in thefamily Epidermoptidae, follicle mites in the family Demodicidae, grainmites in the family Glycyphagidae, ticks in the order Ixodidae. Ixodesscapularis Say (deer tick); I. holocyclus Neumann (Australian paralysistick); Dermacentor variabilis Say (American dog tick); Amblyommaamericanum Linnaeus (lone star tick) and scab and itch mites in thefamilies Psoroptidae, Pyemotidae and Sarcoptidae.

Insect pests of the order Thysanura are of interest, such as Lepismasaccharina Linnaeus (silverfish); Thermobia domestica Packard(firebrat).

Additional arthropod pests covered include: spiders in the order Araneaesuch as Loxosceles reclusa Gertsch and Mulaik (brown recluse spider) andthe Latrodectus mactans Fabricius (black widow spider) and centipedes inthe order Scutigeromorpha such as Scutigera coleoptrata Linnaeus (housecentipede).

Insect pest of interest include the superfamily of stink bugs and otherrelated insects including but not limited to species belonging to thefamily Pentatomidae (Nezara viridula, Halyomorpha halys, Piezodorusguildini, Euschistus servus, Acrosternum hilare, Euschistus heros,Euschistus tristigmus, Acrosternum hilare, Dichelops furcatus, Dichelopsmelacanthus, and Bagrada hilaris (Bagrada Bug)), the family Plataspidae(Megacopta cribraria—Bean plataspid) and the family Cydnidae(Scaptocoris castanea—Root stink bug) and Lepidoptera species includingbut not limited to: diamond-back moth, e.g., Helicoverpa zea Boddie;soybean looper, e.g., Pseudoplusia includens Walker and velvet beancaterpillar e.g., Anticarsia gemmatalis Hubner.

Methods for measuring pesticidal activity are well known in the art.See, for example, Czapla and Lang, (1990) J. Econ. Entomol.83:2480-2485; Andrews, et al., (1988) Biochem. J. 252:199-206; Marrone,et al., (1985) J. of Economic Entomology 78:290-293 and U.S. Pat. No.5,743,477. Generally, the protein is mixed and used in feeding assays.See, for example Marrone, et al., (1985) J. of Economic Entomology78:290-293. Such assays can include contacting plants with one or morepests and determining the plant's ability to survive and/or cause thedeath of the pests.

Nematodes include parasitic nematodes such as root-knot, cyst and lesionnematodes, including Heterodera spp., Meloidogyne spp. and Globoderaspp.; particularly members of the cyst nematodes, including, but notlimited to, Heterodera glycines (soybean cyst nematode); Heteroderaschachtii (beet cyst nematode); Heterodera avenae (cereal cyst nematode)and Globodera rostochiensis and Globodera pailida (potato cystnematodes). Lesion nematodes include Pratylenchus spp.

Seed Treatment

To protect and to enhance yield production and trait technologies, seedtreatment options can provide additional crop plan flexibility and costeffective control against insects, weeds and diseases. Seed material canbe treated, typically surface treated, with a composition comprisingcombinations of chemical or biological herbicides, herbicide safeners,insecticides, fungicides, germination inhibitors and enhancers,nutrients, plant growth regulators and activators, bactericides,nematocides, avicides and/or molluscicides. These compounds aretypically formulated together with further carriers, surfactants orapplication-promoting adjuvants customarily employed in the art offormulation. The coatings may be applied by impregnating propagationmaterial with a liquid formulation or by coating with a combined wet ordry formulation. Examples of the various types of compounds that may beused as seed treatments are provided in The Pesticide Manual: A WorldCompendium, C. D. S. Tomlin Ed., Published by the British CropProduction Council.

Some seed treatments that may be used on crop seed include, but are notlimited to, one or more of abscisic acid, acibenzolar-S-methyl,avermectin, amitrol, azaconazole, azospirillum, azadirachtin,azoxystrobin, Bacillus spp. (including one or more of cereus, firmus,megaterium, pumilis, sphaericus, subtilis and/or thuringiensis species),Bradyrhizobium spp. (including one or more of betae, canariense,elkanii, iriomotense, japonicum, liaonigense, pachyrhizi and/oryuanmingense), captan, carboxin, chitosan, clothianidin, copper,cyazypyr, difenoconazole, etidiazole, fipronil, fludioxonil,fluoxastrobin, fluquinconazole, flurazole, fluxofenim, harpin protein,imazalil, imidacloprid, ipconazole, isoflavenoids,lipo-chitooligosaccharide, mancozeb, manganese, maneb, mefenoxam,metalaxyl, metconazole, myclobutanil, PCNB, penflufen, penicillium,penthiopyrad, permethrine, picoxystrobin, prothioconazole,pyraclostrobin, rynaxypyr, S-metolachlor, saponin, sedaxane, TCMTB,tebuconazole, thiabendazole, thiamethoxam, thiocarb, thiram,tolclofos-methyl, triadimenol, trichoderma, trifloxystrobin,triticonazole and/or zinc. PCNB seed coat refers to EPA RegistrationNumber 00293500419, containing quintozen and terrazole. TCMTB refers to2-(thiocyanomethylthio) benzothiazole.

Seed varieties and seeds with specific transgenic traits may be testedto determine which seed treatment options and application rates maycomplement such varieties and transgenic traits in order to enhanceyield. For example, a variety with good yield potential but head smutsusceptibility may benefit from the use of a seed treatment thatprovides protection against head smut, a variety with good yieldpotential but cyst nematode susceptibility may benefit from the use of aseed treatment that provides protection against cyst nematode, and soon. Likewise, a variety encompassing a transgenic trait conferringinsect resistance may benefit from the second mode of action conferredby the seed treatment, a variety encompassing a transgenic traitconferring herbicide resistance may benefit from a seed treatment with asafener that enhances the plants resistance to that herbicide, etc.Further, the good root establishment and early emergence that resultsfrom the proper use of a seed treatment may result in more efficientnitrogen use, a better ability to withstand drought and an overallincrease in yield potential of a variety or varieties containing acertain trait when combined with a seed treatment.

Methods for Killing an Insect Pest and Controlling an Insect Population

In some embodiments methods are provided for killing an insect pest,comprising contacting the insect pest, either simultaneously orsequentially, with an insecticidally-effective amount of a recombinantIPD101 polypeptide of the disclosure. In some embodiments methods areprovided for killing an insect pest, comprising contacting the insectpest with an insecticidally-effective amount of one or more of arecombinant pesticidal protein of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14,16, 18, 20, 22, 24, 25, 26, 28, 29, 30, 32, 46, 48, 50, 52, 54, 56, 58,and 60, or a variant or insecticidally active fragment thereof.

In some embodiments methods are provided for controlling an insect pestpopulation, comprising contacting the insect pest population, eithersimultaneously or sequentially, with an insecticidally-effective amountof one or more of a recombinant IPD101 polypeptide of the disclosure. Insome embodiments, methods are provided for controlling an insect pestpopulation, comprising contacting the insect pest population with aninsecticidally-effective amount of one or more of a recombinant IPD101polypeptide of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,25, 26, 28, 29, 30, 32, 46, 48, 50, 52, 54, 56, 58, and 60, or a variantor insecticidally active fragment thereof. As used herein, “controllinga pest population” or “controls a pest” refers to any effect on a pestthat results in limiting the damage that the pest causes. Controlling apest includes, but is not limited to, killing the pest, inhibitingdevelopment of the pest, altering fertility or growth of the pest insuch a manner that the pest provides less damage to the plant,decreasing the number of offspring produced, producing less fit pests,producing pests more susceptible to predator attack or deterring thepests from eating the plant.

In some embodiments methods are provided for controlling an insect pestpopulation resistant to a pesticidal protein, comprising contacting theinsect pest population, either simultaneously or sequentially, with aninsecticidally-effective amount of one or more of a recombinant IPD101polypeptide of the disclosure. In some embodiments, methods are providedfor controlling an insect pest population resistant to a pesticidalprotein, comprising contacting the insect pest population with aninsecticidally-effective amount of one or more of a recombinant IPD101polypeptide of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,25, 26, 28, 29, 30, 32, 46, 48, 50, 52, 54, 56, 58, and 60, or a variantor insecticidally active fragment thereof.

In some embodiments methods are provided for protecting a plant from aninsect pest, comprising expressing in the plant or cell thereof at leastone recombinant polynucleotide encoding an IPD101 polypeptide of thedisclosure. In some embodiments methods are provided for protecting aplant from an insect pest, comprising expressing in the plant or cellthereof a recombinant polynucleotide encoding one or more IPD101polypeptides of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,25, 26, 28, 29, 30, 32, 46, 48, 50, 52, 54, 56, 58, and 60, or variantsor insecticidally active fragments thereof.

Insect Resistance Management (IRM) Strategies

Expression of B. thuringiensis δ-endotoxins in transgenic corn plantshas proven to be an effective means of controlling agriculturallyimportant insect pests (Perlak, et al., 1990; 1993). However, in certaininstances insects have evolved that are resistant to B. thuringiensisδ-endotoxins expressed in transgenic plants. Such resistance, should itbecome widespread, would clearly limit the commercial value of germplasmcontaining genes encoding such B. thuringiensis δ-endotoxins.

One way of increasing the effectiveness of the transgenic insecticidesagainst target pests and contemporaneously reducing the development ofinsecticide-resistant pests is to use provide non-transgenic (i.e.,non-insecticidal protein) refuges (a section of non-insecticidalcrops/corn) for use with transgenic crops producing a singleinsecticidal protein active against target pests. The United StatesEnvironmental Protection Agency(epa.gov/oppbppdl/biopesticides/pips/bt_corn_refuge_2006.htm, which canbe accessed using the www prefix) publishes the requirements for usewith transgenic crops producing a single Bt protein active againsttarget pests. In addition, the National Corn Growers Association, ontheir website:(ncga.com/insect-resistance-management-fact-sheet-bt-corn, which can beaccessed using the www prefix) also provides similar guidance regardingrefuge requirements. Due to losses to insects within the refuge area,larger refuges may reduce overall yield.

Another way of increasing the effectiveness of the transgenicinsecticides against target pests and contemporaneously reducing thedevelopment of insecticide-resistant pests would be to have a repositoryof insecticidal genes that are effective against groups of insect pestsand which manifest their effects through different modes of action.

Expression in a plant of two or more insecticidal compositions toxic tothe same insect species, each insecticide being expressed at efficaciouslevels would be another way to achieve control of the development ofresistance. This is based on the principle that evolution of resistanceagainst two separate modes of action is far more unlikely than only one.Roush, for example, outlines two-toxin strategies, also called“pyramiding” or “stacking,” for management of insecticidal transgeniccrops. (The Royal Society. Phil. Trans. R. Soc. Lond. B. (1998)353:1777-1786). Stacking or pyramiding of two different proteins eacheffective against the target pests and with little or nocross-resistance can allow for use of a smaller refuge. The USEnvironmental Protection Agency requires significantly less (generally5%) structured refuge of non-Bt corn be planted than for single traitproducts (generally 20%). There are various ways of providing the IRMeffects of a refuge, including various geometric planting patterns inthe fields and in-bag seed mixtures, as discussed further by Roush.

In some embodiments the IPD101 polypeptides of the disclosure are usefulas an insect resistance management strategy in combination (i.e.,pyramided) with other pesticidal proteins or other transgenes (i.e., anRNAi trait) including but not limited to Bt toxins, Xenorhabdus sp. orPhotorhabdus sp. insecticidal proteins, other insecticidally activeproteins, and the like.

Provided are methods of controlling Lepidoptera and/or Coleoptera insectinfestation(s) in a transgenic plant that promote insect resistancemanagement, comprising expressing in the plant at least two differentinsecticidal proteins having different modes of action.

In some embodiments the methods of controlling Lepidoptera and/orColeoptera insect infestation in a transgenic plant and promoting insectresistance management comprises the presentation of at least one of theIPD101 polypeptide insecticidal proteins to insects in the orderLepidoptera and/or Coleoptera.

In some embodiments the methods of controlling Lepidoptera and/orColeoptera insect infestation in a transgenic plant and promoting insectresistance management comprises the presentation of at least one of theIPD101 polypeptides of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20,22, 24, 25, 26, 28, 29, 30, 32, 46, 48, 50, 52, 54, 56, 58, and 60, orvariants or insecticidally active fragments thereof, insecticidal toinsects in the order Lepidoptera and/or Coleoptera.

In some embodiments the methods of controlling Lepidoptera and/orColeoptera insect infestation in a transgenic plant and promoting insectresistance management comprise expressing in the transgenic plant anIPD101 polypeptide and a Cry protein or other insecticidal protein toinsects in the order Lepidoptera and/or Coleoptera having differentmodes of action.

In some embodiments the methods of controlling Lepidoptera and/orColeoptera insect infestation in a transgenic plant and promoting insectresistance management comprise expression in the transgenic plant of atleast one of an IPD101 polypeptide of SEQ ID NOS: 2, 4, 6, 8, 10, 12,14, 16, 18, 20, 22, 24, 25, 26, 28, 29, 30, 32, 46, 48, 50, 52, 54, 56,58, and 60, or variants or insecticidally active fragments thereof and aCry protein or other insecticidal protein to insects in the orderLepidoptera and/or Coleoptera, where the IPD101 polypeptide and Cryprotein have different modes of action.

Also provided are methods of reducing likelihood of emergence ofLepidoptera and/or Coleoptera insect resistance to transgenic plantsexpressing in the plants insecticidal proteins to control the insectspecies, comprising expression of at least one of an IPD101 polypeptideinsecticidal to the insect species in combination with a secondinsecticidal protein to the insect species having different modes ofaction.

Also provided are means for effective Lepidoptera and/or Coleopterainsect resistance management of transgenic plants, comprisingco-expressing at high levels in the plants two or more insecticidalproteins or other insecticidal transgenes (e.g., an RNAi trait) toxic toLepidoptera and/or Coleoptera insects but each exhibiting a differentmode of effectuating its killing activity, wherein two or more of theinsecticidal proteins or other insecticidal transgenes comprise anIPD101 polypeptide and a Cry protein. Also provided are means foreffective Lepidoptera and/or Coleoptera insect resistance management oftransgenic plants, comprising co-expressing at high levels in the plantstwo or more insecticidal proteins or other insecticidal transgenes(e.g., an RNAi trait) toxic to Lepidoptera and/or Coleoptera insects buteach exhibiting a different mode of effectuating its killing activity,wherein two or more insecticidal proteins or other insecticidaltransgenes comprise at least one of an IPD101 polypeptide of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 25, 26, 28, 29, 30, 32, 46,48, 50, 52, 54, 56, 58, and 60, or variants or insecticidally activefragments thereof and a Cry protein or other insecticidally activeprotein.

In addition, methods are provided for obtaining regulatory approval forplanting or commercialization of plants expressing proteins insecticidalto insects in the order Lepidoptera and/or Coleoptera, comprising thestep of referring to, submitting or relying on insect assay binding datashowing that the IPD101 polypeptide does not compete with binding sitesfor Cry proteins in such insects. In addition, methods are provided forobtaining regulatory approval for planting or commercialization ofplants expressing proteins insecticidal to insects in the orderLepidoptera and/or Coleoptera, comprising the step of referring to,submitting or relying on insect assay binding data showing that one ormore of the IPD101 polypeptides of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14,16, 18, 20, 22, 24, 25, 26, 28, 29, 30, 32, 46, 48, 50, 52, 54, 56, 58,and 60, or variant or insecticidally active fragment thereof does notcompete with binding sites for Cry proteins in such insects.

Methods for Increasing Plant Yield

Methods for increasing plant yield are provided. The methods compriseproviding a plant or plant cell expressing a polynucleotide encoding thepesticidal polypeptide sequence disclosed herein and growing the plantor a seed thereof in a field infested with a pest against which thepolypeptide has pesticidal activity. In some embodiments, thepolypeptide has pesticidal activity against a Lepidopteran, Coleopteran,Dipteran, Hemipteran or nematode pest, and the field is infested with aLepidopteran, Hemipteran, Coleopteran, Dipteran or nematode pest.

As defined herein, the “yield” of the plant refers to the quality and/orquantity of biomass produced by the plant. “Biomass” as used hereinrefers to any measured plant product. An increase in biomass productionis any improvement in the yield of the measured plant product.Increasing plant yield has several commercial applications. For example,increasing plant leaf biomass may increase the yield of leafy vegetablesfor human or animal consumption. Additionally, increasing leaf biomasscan be used to increase production of plant-derived pharmaceutical orindustrial products. An increase in yield can comprise any statisticallysignificant increase including, but not limited to, at least a 1%increase, at least a 3% increase, at least a 5% increase, at least a 10%increase, at least a 20% increase, at least a 30%, at least a 50%, atleast a 70%, at least a 100% or a greater increase in yield compared toa plant not expressing the pesticidal sequence.

In specific methods, plant yield is increased as a result of improvedpest resistance of a plant expressing at least one IPD101 polypeptidedisclosed herein. Expression of the IPD101 polypeptide(s) results in areduced ability of a pest to infest or feed on the plant, thus improvingplant yield.

Methods of Processing

Further provided are methods of processing a plant, plant part or seedto obtain a food or feed product from a plant, plant part or seedcomprising at least one IPD101 polynucleotide. The plants, plant partsor seeds provided herein, can be processed to yield oil, proteinproducts and/or by-products that are derivatives obtained by processingthat have commercial value. Non-limiting examples include transgenicseeds comprising a nucleic acid molecule encoding one or more IPD101polypeptides which can be processed to yield soy oil, soy productsand/or soy by-products.

“Processing” refers to any physical and chemical methods used to obtainany soy product and includes, but is not limited to, heat conditioning,flaking and grinding, extrusion, solvent extraction or aqueous soakingand extraction of whole or partial seeds

The following examples are offered by way of illustration and not by wayof limitation.

EXAMPLES Example 1—Identification of an Insecticidal Protein ActiveAgainst Western Corn Rootworm (WCRW) from Strain JH70371-1

The insecticidal protein IPD101Aa was identified by proteinpurification, N-terminal amino acid sequencing, and PCR cloning frombacterial strain JH70371-1 as follows. Insecticidal activity againstWCRW was observed from a cell lysate of strain JH70371-1 that was grownin Terrific Broth (BD Difco™, Catalog #243820) and cultured overnight at28° C. with shaking at 200 rpm. This insecticidal activity exhibitedheat and protease sensitivity indicating a proteinaceous nature.

Bioassays with WCRW were conducted using the cell lysate samples mixedwith molten low-melt WCRW diet (Frontier Agricultural Sciences, Newark,Del.) in a 96 well format. WCRW neonates were placed into each well of a96 well plate. The assay was run four days at 25° C. and then was scoredfor insect mortality and stunting of insect growth. The scores werenoted as dead (3), severely stunted (2) (little or no growth but alive),stunted (1) (growth to second instar but not equivalent to controls) orno observed activity (0). Samples demonstrating mortality or severestunting were further studied.

Genomic DNA of isolated strain JH70371-1 was prepared according to alibrary construction protocol and sequenced using the Illumina® GenomeAnalyzer IIx (Illumina Inc., San Diego, Calif.). The nucleic acid contigsequences were assembled and open reading frames were generated. The 16Sribosomal DNA sequence of strain JH70371-1 was BLAST searched againstthe NCBI database which indicated that this is a Lysinibacillus sp.

Cell pellets of strain JH70371-1 were homogenized at −30,000 psi afterre-suspension in 20 mM MOPS buffer, pH 7 with “Complete, EDTA-free”protease inhibitor cocktail (Roche, Indianapolis, Ind.). The crudelysate was cleared by centrifugation and desalted into 20 mM Tris, pH8.5 using a HiPrep™ 26/10 desalting column (GE Healthcare, Piscataway,N.J.) and then loaded onto a CaptoQ™ column (GE Healthcare, Piscataway,N.J.) equilibrated in 20 mM Tris, pH 8.5 and eluted with a gradient of 0to 0.4 M NaCl over 30 column volumes (CV). Active fractions were pooledand loaded onto a Superdex™ 200 column (GE Healthcare) equilibrated in100 mM ammonium bicarbonate. SDS-PAGE analysis of fractions indicatedthat WCRW activity coincided with a prominent protein band afterstaining with GelCode® Blue Stain Reagent (Thermo Fisher Scientific®).The protein band was excised, digested with trypsin and analyzed bynano-liquid chromatography/electrospray tandem mass spectrometry(nano-LC/ESI-MS/MS) on a Thermo Q Exactive™ Orbitrap™ mass spectrometer(Thermo Fisher Scientific®, 81 Wyman Street, Waltham, Mass. 02454)interfaced with an Eksigent NanoLC 1-D Plus nano-lc system (AB Sciex™,500 Old Connecticut Path, Framingham, Mass. 01701). Proteinidentification was done by database searches using Mascot® (MatrixScience, 10 Perrins Lane, London NW3 1QY UK). The searches against anin-house database and NCBI non-redundant database (nr) identified thenovel polypeptide IPD101Aa (SEQ ID NO: 2) which is encoded by thepolynucleotide of SEQ ID NO: 1. Cloning and recombinant expressionconfirmed the insecticidal activity of the IPD101Aa against WCRW.

Example 2—Identification of Homologs of IPD101Aa

In addition to presence in strain JH70371-1, BLAST searches identifiedseveral homologs having varying percent amino acid identity to IPD101Aa(SEQ ID NO: 2): IPD101Ab (SEQ ID NO: 4) with 98.2% identity and 99.7%similarity to IPD101Aa was identified in DuPont Pioneer strainPMCH4031E7-1. IPD101Ac (SEQ ID NO: 6) with 97.9% identity and 99.4%similarity to IPD101Aa was identified in DuPont Pioneer strainPMCH4053D11b. IPD101Ba (SEQ ID NO: 8) with 80.9% identity and 89.7%similarity to IPD101Aa was identified in the public NCBI database asgi_928971774_ref_WP_053996211 as a hypothetical protein fromLysinibacillus macroides. IPD101Ca (SEQ ID NO: 10) with 77.0% identityand 87.9% similarity to IPD101Aa was identified in the public NCBIdatabase as gi_499133538_ref_WP_010861479 as a hypothetical protein fromLysinibacillus sphaericus. In addition, IPD101Cb (SEQ ID NO: 12) wasidentified in DuPont Pioneer strain AM2685 with 78.2% identity toIPD101Aa. IPD101Cc (SEQ ID NO: 14) with 88.2% identity to IPD101Aa wasidentified in DuPont Pioneer strain JAPH0723-1. IPD101Cd (SEQ ID NO: 16)with 73.0% identity to IPD101Aa was identified in DuPont Pioneer strainAM11987. IPD101Ce (SEQ ID NO: 18) with 69.4% identity to IPD101Aa wasidentified in DuPont Pioneer strain DP3525M. IPD101Cf (SEQ ID NO: 20)with 78.8% identity to IPD101Aa was identified in DuPont Pioneer strainBD22. IPD101Ea (SEQ ID NO: 22) with 54.1% identity to IPD101Aa wasidentified in the public NCBI database as WP_024363526.1 as ahypothetical protein from Lysinibacillus sphaericus. IPD101Eb (SEQ IDNO: 24) with 53.5% identity to IPD101Aa was identified in the publicNCBI database as AHN24097.1 as a hypothetical protein fromLysinibacillus varians. IPD101Ee (SEQ ID NO: 25) with 55.4% identity toIPD101Aa was identified in the public NCBI database as WP_058336899 as ahypothetical protein from Bacillus sp. IPD101Fa (SEQ ID NO: 26) with45.0% identity to IPD101Aa was identified in the public NCBI database asWP_047474321 as a hypothetical protein from Bacillus amyloliquefaciens.IPD101Fb (SEQ ID NO: 28) with 44.6% identity to IPD101Aa was identifiedin DuPont Pioneer strain PMC4018E9-1. IPD101Ga (SEQ ID NO: 29) with33.7% identity to IPD101Aa was identified in the public NCBI database asWP_050637303 as a hypothetical protein from Candidatus stoquefichus.IPD101Gb (SEQ ID NO: 30) with 37.8% identity to IPD101Aa was identifiedin the public NCBI database as WP_050637304 as a hypothetical proteinfrom Candidatus stoquefichus. IPD101Gc (SEQ ID NO: 32) with 32.3%identity to IPD101Aa was identified in the public NCBI database asAL041133 as a hypothetical protein from Pseudoalteromonas phenolica.IPD101Gd (SEQ ID NO: 56) with 34.8% identity to IPD101Aa was identifiedin the public NCBI database as WP_066332372 as a hypothetical proteinfrom Flavobacterium crassostreae. IPD101Ge (SEQ ID NO: 58) with 35.1%identity to IPD101Aa was identified in the public NCBI database asWP_066758778 as a hypothetical protein from Chryseobacterium sp.IPD101Gf (SEQ ID NO: 60) with 33.7% identity to IPD101Aa was identifiedin the public NCBI database as WP_063304516 as a hypothetical proteinfrom Pseudovibrio sp. The IPD101Aa homologs and the source of thesequence they were identified from are shown in Table 1.

TABLE 1 Gene Name Source Organism DNA Seq AA seq IPD101Aa JH70371Lysinibacillus sp. SEQ ID NO: 1 SEQ ID NO: 2 IPD101Ab PMCH4031E7-1Lysinibacillus sp. SEQ ID NO: 3 SEQ ID NO: 4 IPD101Ac PMCH4053D11bLysinibacillus sp. SEQ ID NO: 5 SEQ ID NO: 6 IPD101Ba NCBI WP_053996211Lysinibacillus SEQ ID NO: 7 SEQ ID NO: 8 macroides IPD101Ca NCBIWP_010861479.1 Lysinibacillus SEQ ID NO: 9 SEQ ID NO: 10 sphaericusIPD101Cb AM2685 Lysinibacillus sp. SEQ ID NO: 11 SEQ ID NO: 12 IPD101CcJAPH0723 Lysinibacillus sp. SEQ ID NO: 13 SEQ ID NO: 14 IPD101Cd AM11987Lysinibacillus sp. SEQ ID NO: 15 SEQ ID NO: 16 IPD101Ce DP3525M Bacillussp. SEQ ID NO: 17 SEQ ID NO: 18 IPD101Cf BD22 Lysinibacillus sp. SEQ IDNO: 19 SEQ ID NO: 20 IPD101Ea NCBI WP_024363526.1 Lysinibacillus SEQ IDNO: 21 SEQ ID NO: 22 sphaericus IPD101Eb NCBI AHN24097.1 Lysinibacillusvarians SEQ ID NO: 23 SEQ ID NO: 24 IPD101Ee NCBI WP_058336899 Bacillussp. SEQ ID NO: 25 IPD101Fa NCBI WP_047474321 Bacillus SEQ ID NO: 26amyloliquefaciens IPD101Fb PMC4018E9-1 Pseudomonas SEQ ID NO: 27 SEQ IDNO: 28 monteilii IPD101Ga NCBI WP_050637303 Candidatus SEQ ID NO: 29stoquefichus IPD101Gb NCBI WP_050637304 Candidatus SEQ ID NO: 30stoquefichus IPD101Gc NCBI AL041133 Pseudoalteromonas SEQ ID NO: 31 SEQID NO: 32 phenolica IPD101Gd WP_066332372 Flavobacterium SEQ ID NO: 55SEQ ID NO: 56 crassostreae IPD101Ge WP_066758778 Chryseobacterium sp.SEQ ID NO: 57 SEQ ID NO: 58 IPD101Gf WP_063304516 Pseudovibrio sp. SEQID NO: 59 SEQ ID NO: 60

The amino acid sequence identities of the IPD101Aa homologs using theNeedlemann-Wunsch algorithm, calculated with a Gap creation penalty: 8and Gap extension penalty: 2, are shown in Table 2.

IPD101Ab IPD101Ac IPD101Ba IPD101Ca IPD101Cb IPD101Cc IPD101Cd IPD101CeIPD101Cf IPD101Ea IPD101Aa 98.2 97.9 80.9 77.0 78.2 78.8 73.3 69.4 78.854.1 IPD101Ab — 97.9 80.9 76.4 77.6 78.2 73.0 69.7 78.2 53.8 IPD101Ac —— 81.2 75.8 77.3 77.9 72.4 69.1 77.6 53.8 IPD101Ba — — — 82.1 82.7 82.176.7 72.7 80.6 56.3 IPD101Ca — — — — 92.7 93.3 81.5 75.5 90.6 58.1IPD101Cb — — — — — 97.0 82.4 74.5 88.2 60.1 IPD101Cc — — — — — — 81.275.4 88.5 59.0 IPD101Cd — — — — — — — 71.1 80.6 59.4 IPD101Ce — — — — —— — — 75.5 56.8 IPD101Cf — — — — — — — — — 57.7 IPD101Ea — — — — — — — —— — IPD101Eb — — — — — — — — — — IPD101Ee — — — — — — — — — — IPD101Fa —— — — — — — — — — IPD101Fb — — — — — — — — — — IPD101Ga — — — — — — — —— — IPD101Gb — — — — — — — — — — IPD101Gc — — — — — — — — — — IPD101Gd —— — — — — — — — — IPD101Ge — — — — — — — — — — IPD101Gf — — — — — — — —— — IPD101Eb IPD101Ee IPD101Fa IPD101Fb IPD101Ga IPD101Gb IPD101GcIPD101Gd IPD101Ge IPD101Gf IPD101Aa 53.5 55.4 45.0 44.6 33.7 37.8 32.336.8 36.0 35.8 IPD101Ab 53.2 56.5 45.5 44.6 34.3 38.1 32.0 37.1 36.335.6 IPD101Ac 53.2 55.4 45.0 44.4 33.7 38.5 32.0 37.1 36.3 35.6 IPD101Ba55.7 54.6 45.5 44.9 34.9 35.8 30.5 38.0 38.0 35.6 IPD101Ca 58.1 55.246.3 43.9 33.4 37.6 30.2 38.7 37.6 36.0 IPD101Cb 59.8 54.9 45.8 44.632.9 36.6 29.9 39.8 37.0 36.0 IPD101Cc 58.7 53.8 45.5 45.6 33.4 36.329.8 38.8 37.0 36.3 IPD101Cd 58.8 56.9 46.3 42.7 32.8 34.6 29.3 37.236.3 34.7 IPD101Ce 56.5 55.2 48.2 43.2 33.1 37.8 31.7 38.0 35.6 35.3IPD101Cf 57.4 55.5 44.4 45.0 34.6 38.3 30.2 38.2 36.6 37.1 IPD101Ea 99.451.4 46.5 40.9 31.3 35.0 31.6 35.7 34.3 32.2 IPD101Eb — 51.2 46.2 40.631.3 34.7 31.2 35.7 33.7 32.0 IPD101Ee — — 48.4 36.6 31.7 33.2 28.2 33.831.9 30.2 IPD101Fa — — — 33.4 31.0 35.2 29.2 30.5 30.6 30.3 IPD101Fb — —— — 31.5 33.0 28.7 35.6 35.5 36.8 IPD101Ga — — — — — 38.2 29.6 29.7 28.233.4 IPD101Gb — — — — — — 29.2 32.8 36.2 31.2 IPD101Gc — — — — — — —28.4 27.9 29.4 IPD101Gd — — — — — — — — 78.6 30.7 IPD101Ge — — — — — — —— — 33.1 IPD101Gf — — — — — — — — — —

Example 3—Cloning and Expression of IPD101Aa in E. coli

An open reading frame containing the IPD101Aa coding sequence wasidentified in the genomic sequence of stain JH70371 using peptidefragments from MS analysis. This sequence was used to design thefollowing primers, AAAGGATCCATGCATACAACAATTGATATTGATCT (IPD101Aa For)(SEQ ID NO: 33) and TTTCTCGAGCTATTTTTTAAATGCACGAGC (IPD101Aa Rev) (SEQID NO: 34), to subclone the IPD101Aa coding sequence into the pET-28avector (Novagen) using the BamHI/XhoI restriction sites in frame with anN-terminal 6×-His tag and the IPD101Aa native stop codon (TAG). The KODHot Start Master Mix (EMD Biosciences, San Diego, Calif.) was used forPCR amplification of the IPD101Aa gene on a BioRad C1000 Touch thermalcycler. Amplicons were gel purified, ligated (T4 DNA Ligase, New EnglandBioLabs, Ipswich, Mass.) into the BamHI/XhoI digested pET28a,transformed into E. coli TOP10 high efficiency chemically competentcells (Invitrogen) and clones were confirmed by sequencing.

The IPD101Aa N-terminal 6×-His tagged construct was transformed intochemically competent BL21 (DE3) cells (Invitrogen) and grown overnightat 37° C. with kanamycin selection and then inoculated to a fresh 2×YTmedium (1:100) and further grown to an optical density of about 0.8-1.2.Protein expression was induced by adding 1.0 mM IPTG and cells werefurther grown at 16° C. for 16 hours. The E. coli expressed proteinswere purified by immobilized metal ion chromatography (IMAC) using TalonCobalt resin (Clonetech: Mountain View, Calif.) according to themanufacturer's protocols. The purified 1.5 mL fractions eluted in 250 mMimidazole were dialyzed into PBS buffer using 6K MWCO Flextubes (IBI:Peosta, Iowa) overnight on a stir plate at 4° C. The dialyzed proteinwas run in diet assays to evaluate the insecticidal protein effects onlarvae of a diversity of Lepidoptera and Coleoptera. Purified anddesalted IPD101Aa N-terminal 6×-His tagged protein was submitted tobioassay against WCRW and was found to be active as shown in Table 4below.

Example 4—Cloning of IPD101Aa Homologs IPD101Cb, Cc, Cd, Ce and Cf

Genes with sequence similarity to the polynucleotide sequence forIPD101Aa (SEQ ID NO: 1) identified from internal databases were PCRamplified from DNA prepared from the source organism (Table 1) using theprimers designed to the coding sequences of each homolog (Table 3). Allprimers contained greater than 30 nucleotides of homology to pET28a(Novagen) or a modified version of pET28a. The PCR products were gelpurified, assembled using the Gibson Assembly Cloning Kit (New EnglandBiolabs, Ipswich, Mass.) with the expression vectors having the matchingoverlap sequence, transformed into E. coli TOP10 high efficiencychemically competent cells (Invitrogen) and clones were confirmed bysequencing. Purified and desalted IPD101 N-terminal 6×-His taggedhomolog protein was submitted to bioassay against WCRW and was observedto have activity as referenced below (Table 4 below).

TABLE 3 PCR primers used to clone homologs of IPD101Aa. Forward ReversePrimer Primer Gene Name SEQ ID Forward Primer SEQ ID Reverse PrimerIPD101Cb SEQ ID ACTGGTGGACAGCAAA SEQ ID CTCGAGTGCGGCCGCAAGC NO: 43TGGGTCGCGGATCCATG NO: 44 TTTTAGGCTTTAAATGCTCG CAMACTACAATTGATATGCAACGTAATA TCGATCTTAA IPD101Cc SEQ ID ACTGGTGGACAGCAAA SEQ IDCTCGAGTGCGGCCGCAAGC NO: 41 TGGGTCGCGGATCCATG NO: 42 TTTTATGCTTTAAATGCTCGCAMACTACAATTGATA TGCTACGTAGTA TCGATCTTAA IPD101Cd SEQ IDACTGGTGGACAGCAAA SEQ ID CTCGAGTGCGGCCGCAAGC NO: 37 TGGGTCGCGGATCCATGNO: 38 TTCTATGCTTTATATGCGCG CAMACTACAATTGATA TGCTACATAATA TCGATCTTAAIPD101Ce SEQ ID CCGCGCGGCAGCATCG SEQ ID CTTTCGACTGAGCCTTTCGT NO: 39AGGGAAGGCATATGCA NO: 40 TTTACTCGAGTTATGATCGA AATTKCACATGATATTGTATGCACGAGCAACGTAGT ATTTAAGG A IPD101Cf SEQ ID ACTGGTGGACAGCAAA SEQ IDCTCGAGTGCGGCCGCAAGC NO: 35 TGGGTCGCGGATCCATG NO: 36 TTTTAAGCTTTATATGCTCGCAMACTACAATTGATA TGCTACGTAATA TCGATCTTAA

Example 5—Cloning of IPD101Aa Homologs IPD101Ca, Ea, and Eb

The IPD101Ca, IPD101Ea, and IPD101Eb amino acid sequences wereidentified by a BLAST search of the public non-redundant proteinsequence database (Table 1). The corresponding coding sequences (SEQ IDNO: 9, SEQ ID NO: 21, and SEQ ID NO: 23, respectively) were generated assynthetic DNA fragments with BamHI/XhoI restriction sites, ligated intopET28a (Novagen) digested with BamHI/XhoI, transformed into E. coliTOP10 high efficiency chemically competent cells (Invitrogen), andconfirmed by sequencing. Purified and desalted IPD101 N-terminal 6×-Histagged homolog protein was submitted to bioassay against WCRW, andactivity results are presented below (Table 4 below).

TABLE 4 Assay Protein Top_Dose type WCRW FAW CEW ECB SBL BCW VBC SCRWIPD101Aa 1200 ppm incorp Yes No Yes No No No Yes Yes IPD101Ca 1500 ppmincorp Yes No Yes Yes Yes No No NT IPD101Cb 333 ppm incorp Yes NT NT NTNT NT NT NT IPD101Cc 1199 ppm incorp Yes NT NT NT NT NT NT NT IPD101Cd453 ppm incorp No NT NT NT NT NT NT NT IPD101Ce 156 ppm incorp Yes NT NTNT NT NT NT NT IPD101Cf 409 ppm incorp Yes NT NT NT NT NT NT NT IPD101Ea1125 μg/cm² overlay No No No No No No No NT IPD101Eb 20 μg/cm² overlayNo No No No No No No NT “NT” denotes not tested; “WCRW” denotes WesternCorn Rootworm; “FAW” denotes Fall Armyworm; “CEW” denotes Corn Earworm;“ECB” denotes Eastern Corn Borer; “SBL” denotes Soybean Looper; “BCW”denotes Black Cutworm; “VBC” denotes Velvet Bean Caterpillar; “SCRW”denotes Southern Corn Rootworm.

Example 6—Chimeras Between IPD101 Homologs

To generate active variants with diversified sequences, chimeras betweenIPD101Aa (SEQ ID NO: 2) and IPD101Cc (SEQ ID NO: 14) polypeptides weregenerated by multi-PCR fragment overlap assembly. A total of fivechimeras between IPD101Aa and IPD101Cc were constructed and cloned intopET28a with an N-terminal 6× histidine tag as described in Example 4.Constructs were transformed into BL21 DE3 and cultured for proteinexpression. Cell lysates were generated using B-PER® Protein ExtractionReagent from Thermo Scientific (3747 N. Meridian Rd., Rockford, Ill. USA61101) and screened for WCRW insecticidal activity. Table 5 shows thechimera boundaries and the % sequence identity to IPD101Aa (SEQ ID NO:2) as calculated using the Needlemann-Wunsch algorithm with a Gapcreation penalty: 8 and Gap extension penalty: 2.

TABLE 5 Percent sequence identity of chimeras to IPD101Aa. % Seq.identity to IPD101Aa WCRW Chimera Designation Polynucleotide (SEQ ID NO:2) active Chimera 23 SEQ ID NO: 45 97 Yes SEQ ID NO: 46 Chimera 27 SEQID NO: 47 90 Yes SEQ ID NO: 48 Chimera 29 SEQ ID NO: 49 95 Yes SEQ IDNO: 50 Chimera 41 SEQ ID NO: 51 87 Yes SEQ ID NO: 52 Chimera 44 SEQ IDNO: 53 82 Yes SEQ ID NO: 54

Example 7—Diet-Based Bioassays with Corn Rootworm for Determination ofLC50 and IC50

Standardized corn rootworm diet incorporation bioassays similar to Zhao,J.-Z. et al. (J. Econ. Entomol. 109: 1369-1377 (2016)) were utilized totest the activity of the IPD101Aa polypeptide (SEQ ID NO: 2) againstWCRW. Corn rootworm diet was prepared according to manufacturer'sguideline for Diabrotica diet (Frontier, Newark, Del.). The testinvolved six different IPD101Aa polypeptide doses plus buffer controlwith 32 observations for each dose in each bioassay. Neonates wereinfested into 96-well plates containing a mixture of the IPD101Aapolypeptide (5 μL/well) and diet (25 μL/well), each well withapproximately 5 to 8 larvae (<24 h post hatch). After one day a singlelarva was transferred into each well of a second 96-well platecontaining a mixture of the IPD101Aa polypeptide (20 μL/well) and diet(100 μL/well) at the same concentration as the treatment to which theinsect was exposed on the first day. For NCRW assays, two neonates wereinfested directly into each well of a 96-well plate containing a mixtureof the IPD101Aa polypeptide (20 μL/well) and diet (100 μL/well).

The plates were incubated at 27° C., 65% RH in the dark for 6 days. Theplates with a single WCRW larva per well were scored as dead, severelystunted (>60% reduction in size compared to control larvae) or notaffected. The plates infested with two NCRW larvae per well were scoredbased on the least affected individual for each well. The mortality datawere analyzed by the PROBIT procedure in SAS software (Version 9.4, SASInstitute. Cary, N.C., USA) to determine the lethal concentrationsaffecting 50% of larvae (LC₅₀). Similarly, the total numbers of dead andseverely stunted larvae were used to calculate the growth inhibitionconcentrations affecting 50% of the larvae (IC₅₀).

The LC50 and IC50 against WCRW (Diabrotica virgifera virgifera) were 5.1ppm and 3.0 ppm, respectively and against NCRW (Diabrotica barberi) were54.2 ppm and 11.6 ppm, respectively. The results are shown in Table 6.

TABLE 6 Diet-based bioassays of IPD101 Aa on WCRW and NCRW. N-6xHisIPD101Aa Insect LC/IC (μg/mL, 6 d) 95% CL Slope N WCRW* LC50 5.1 3.3-7.22.2 159 IC50 3.0 2.1-3.9 3.6 127 NCRW** LC50 54.2 41.5-68.8 2.5 244 IC5011.6  7.3-14.0 4.3 212 *One larva per well method; **Two larvae per wellmethod.

Example 8—Mode of Action

Bioactivity of purified recombinant protein incorporated into artificialdiet revealed toxicity of IPD101Aa (SEQ ID NO: 2) to WCRW larvae. Tounderstand the mechanism of IPD101Aa toxicity, specific binding of thepurified protein with WCRW midgut tissue was evaluated by in vitrocompetition assays. Midguts were isolated from third instar WCRW larvaeto prepare brush border membrane vesicles (BBMV) following a methodmodified from Wolfersberger et al. (Comp Bioch Physiol 86A: 301-308(1987)) using amino-peptidase activity to track enrichment. BBMVsrepresent the apical membrane component of the epithelial cell lining ofinsect midgut tissue and therefore serve as a model system for howinsecticidal proteins interact within the gut following ingestion.

Recombinant IPD101Aa was expressed and purified from an E. coliexpression system utilizing a carboxy-terminal poly-histidine fusion tag(6×His). The full length purified protein (SEQ ID NO: 2) was labeledwith Alexa-Fluor® 488 (Life Technologies) and unincorporated fluorophorewas separated from labeled protein using buffer exchange resin (LifeTechnologies, A30006) following manufacturer's recommendations. Prior tobinding experiments, proteins were quantified by gel densitometryfollowing Simply Blue® (Thermo Scientific) staining of SDS-PAGE resolvedsamples that included BSA as a standard.

Binding buffer consisted of PBS supplemented with 0.1% of Tween 20, pH7.4. To demonstrate specific binding and to evaluate affinity, BBMVs (1μg) were incubated with Alexa-labeled IPD101Aa (1.5 nM) in 100 μL ofBinding buffer for 1 h at RT in the absence and presence of increasingconcentrations of unlabeled IPD101Aa. Centrifugation at 20,000×g wasused to pellet the BBMVs to separate unbound toxin remaining insolution. The BBMV pellet was then washed twice with Binding buffer toeliminate remaining unbound toxin. The final BBMV pellet (with boundfluorescent toxin) was solubilized in reducing Laemmli sample buffer,heated to 100° C. for 5 minutes, and subjected to SDS-PAGE using 4-12%Bis-Tris polyacrylamide gels (Life Technologies). The amount ofAlexa-labeled IPD101Aa in the gel from each sample was measured by adigital fluorescence imaging system (Image Quant LAS4000 GE Healthcare).Digitized images were analyzed by densitometry software (Phoretix 1D,TotalLab, Ltd.).

The apparent affinity of IPD101Aa for WCRW BBMVs was estimated based onthe concentration of unlabeled protein that was needed to reduce thebinding of Alexa-labeled IPD101Aa by 50% (EC50 value). This value wasapproximately 2 nM for IPD101Aa binding with WCR BBMVs (FIG. 2).

The above description of various illustrated embodiments of thedisclosure is not intended to be exhaustive or to limit the scope to theprecise form disclosed. While specific embodiments of and examples aredescribed herein for illustrative purposes, various equivalentmodifications are possible within the scope of the disclosure, as thoseskilled in the relevant art will recognize. The teachings providedherein can be applied to other purposes, other than the examplesdescribed above. Numerous modifications and variations are possible inlight of the above teachings and, therefore, are within the scope of theappended claims.

These and other changes may be made in light of the above detaileddescription. In general, in the following claims, the terms used shouldnot be construed to limit the scope to the specific embodimentsdisclosed in the specification and the claims.

The entire disclosure of each document cited (including patents, patentapplications, journal articles, abstracts, manuals, books or otherdisclosures) in the Background, Detailed Description, and Examples isherein incorporated by reference in their entireties.

Efforts have been made to ensure accuracy with respect to the numbersused (e.g. amounts, temperature, concentrations, etc.) but someexperimental errors and deviations should be allowed for. Unlessotherwise indicated, parts are parts by weight, molecular weight isaverage molecular weight; temperature is in degrees centigrade; andpressure is at or near atmospheric.

1-8. (canceled)
 9. A DNA construct comprising a heterologous regulatoryelement and a polynucleotide encoding a polypeptide having at least 95%sequence identity to the amino acid sequence of SEQ ID NO:
 12. 10. Atransgenic plant or plant cell comprising the DNA construct of claim 9.11. (canceled)
 12. (canceled)
 13. A method for controlling an insectpest population, comprising contacting the insect pest population with apolypeptide having at least 95% sequence identity to the amino acidsequence of SEQ ID NO:
 12. 14. A method of inhibiting growth or killingan insect pest, comprising contacting the insect pest with a compositioncomprising a polypeptide having at least 95% sequence identity to theamino acid sequence of SEQ ID NO: 12, O: 50, SEQ ID NO: 52, SEQ ID NO:54, SEQ ID NO: 56, SEQ ID NO: 58, or SEQ ID NO:
 60. 15. The method ofinhibiting growth or killing an insect pest of claim 14, wherein theinsect pest is a Lepidoptera and/or Coleoptera insect pest.
 16. A methodfor controlling an insect pest population, comprising contacting theinsect pest population with the transgenic plant or plant cell of claim10.
 17. A method of inhibiting growth or killing an insect pest,comprising transforming a plant with the DNA construct of claim
 9. 18.The method of claim 17, further comprising contacting the insect pestwith the transgenic plant or plant cell.
 19. The method of claim 18,wherein the insect pest is Western Corn Rootworm (Diabrotica virgiferavirgifera).
 20. The method of claim 14, wherein the insect pest orinsect pest population is resistant to at least one Bt toxin. 21.(canceled)